CN104583387B

CN104583387B - Transformant of Schizosaccharomyces Pombe mutant, and cloning vector

Info

Publication number: CN104583387B
Application number: CN201380044077.2A
Authority: CN
Inventors: 小林真弓; 东田英毅; 木村修郎; 木村修一郎; 禅院进; 原太志
Original assignee: Asahi Glass Co Ltd
Current assignee: AGC Inc
Priority date: 2012-08-20
Filing date: 2013-08-20
Publication date: 2017-05-03
Anticipated expiration: 2033-08-20
Also published as: EP2886642A4; JP6206408B2; EP2886642A1; EP2886642B1; US20150240250A1; JPWO2014030644A1; CN104583387A; US9765347B2; WO2014030644A1

Abstract

Provided are: a transformant of a Schizosaccharomyces Pombe (S. Pombe) mutant with which beta-glucosidase can be produced and collected without requiring a complicated isolation step; and a vector useful for transforming Schizosaccharomyces-group yeast. The transformant of an S. Pombe mutant exhibits increased Gsf activity, reduced or no pyruvyl transferase (Pvg 1) enzyme activity, and is characterized by having, in a chromosome or as an extrachromosomal gene, a promoter sequence and a terminator sequence for expressing a structural gene sequence for coding beta-glucosidase derived from a filimentous fungus, and said structural genes. Furthermore, the present invention relates to a cloning vector, expression vector, transformant, etc., useful for transforming Schizosaccharomyces-group yeast, characterized by having a promoter having the hsp9 gene or a promoter having the ihc1 gene

Description

The transformant and cloning vector of schizosaccharomyces pombe mutant

Technical field

The present invention relates to have asexual flocculability, the schizosaccharomyces pombe of β-glucosyl enzym can be produced The transformant of (Schizosaccharomyces pombe) mutant, and the load useful to the conversion of fragmentation Saccharomyces cerevisiae Body.

Background technology

In order to be produced the sugar as fermentation raw material, Jin Ersheng by cellulose series biomass such as timber, straw, rice husk, weeds The biomass fuels such as generation ethanol, need to decompose the cellulose of the main composition as plant cell wall.It is fine The decomposition of dimension element has the acid mashing system such as concentrated sulfuric acid mashing system, dilute sulfuric acid mashing system, using the method such as enzymatic conversion method of enzyme, in recent years Under carrying out the prosperous background of biotechnology, the research and development of a large amount of enzymatic conversion methods have been carried out.

The enzymatic conversion of cellulose utilizes the class of enzymes of collectively referred to as cellulase.First, with random cutting fibre element chain The amorphous regions of endoglucanase (EG) decomposition of cellulose of activity, make glucose ends exposed.Using cellobiose water Solution enzyme (CBH) decomposes exposed glucose end, makes cellobiose dissociate.Then, decompose free using β-glucosyl enzym (BGL) Cellobiose so that glucose is free.

Due to producing various cellulases and hemicellulase for the avicel cellulose that decomposes, is saccharified, and can be by These enzymes are secreted in a large number to extracellular, therefore the enzymatic conversion of cellulose widely uses the filamentous fungi of aspergillus, trichoderma etc..

Additionally, also having attempted making the cellulase of these filamentous fungis express in different strain, disclose in non-patent literature 1 By the β-glucosyl enzym I for encoding a kind of microorganism Aspergillus aculeatus (Aspergillus aculeatus) as filamentous fungi (BGLI) gene makes these enzymes converting budding yeast (Saccharomyces cerevisiae) in the transformant for obtaining Expression.

But, in the case of enzymatic conversion method, if carrying out the hydrolysis of cellulose by enzyme, glucose is put aside anti- The grape Glyco inhabiting β-glucosyl enzym in system, then put aside, cellobiose savings are answered, and then the cellobiose put aside suppresses inscribe Dextranase and cellobiohydrolase, its result is there is a problem of that cellulose can not decompose completely.For this reason it would be desirable to β-glucose The multifunction of glycosides enzyme.

On the other hand, compared with aspergillus and trichoderma, the genetic analysis of Schizosaccharomyces yeast deepens continuously, and establishes Various useful mutant and channel genes carriers, have the advantages that to be adapted to a large amount of production protein of industry.But, split Grow Saccharomyces cerevisiae and there is no intrinsic β-glucosyl enzym gene, it is impossible to assimilate cellobiose.The present inventor uses coding β-glucose The genetic transformation Schizosaccharomyces yeast of glycosides enzyme, makes these expression of enzymes (patent document 1) in the transformant for obtaining.

In addition, in non-patent literature 1, the glucose suppression of the β-glucosyl enzym for being produced without reference to budding yeast completely System.

On the other hand, in order to reclaim the β-glucosyl enzym secreted by the transformant, isolation medium supernatant is needed The operation of thalline.As separation circuit, the operations such as centrifugation, continuous centrifugal separation, UF membrane can be enumerated, any one is numerous Miscellaneous operation, needs substantial amounts of manpower and time.

Further, with the production-scale expansion of β-glucosyl enzym, be not difficult envision the separation circuit complex increasing Greatly.

On the other hand, because the yeast with asexual flocculability (property asexually flocculated) can easily from training Yeast thalline and nutrient solution that the nutrient solution after terminating isolates flocculation are supported, therefore preferably will tool in the production of β-glucosyl enzym There is the yeast of asexual flocculability as host.

As the yeast with asexual flocculability, it is known that the FLO in budding yeast Saccharomyces cerevisiae Mutant.Additionally, also reporting in fission yeast Schizosaccharomyces pombe (being also referred to as schizosaccharomyces pombe below) Mutant with asexual flocculability (for example, referring to patent document 2).

And on the other hand, the Schizosaccharomyces yeast headed by schizosaccharomyces pombe is and budding yeast Saccharomyces cerevisiae diverse yeast in evolutionary system.Known chromosome structure, genome duplication Mechanism, RNA splicing mechanisms, transcription mechanism, each mechanism such as posttranslational modification and other yeast difference are larger, one part and animal Cell is similar to.Therefore it is widely used as Eukaryotic model (with reference to non-patent literature 2).

Schizosaccharomyces pombe is due to its various features, therefore the single celled eukaryotic being considered closer to higher animal cells is given birth to Thing, it is considered to be as foreign gene, especially from higher mammal gene the expression highly useful yeast of host. Know the expression for being especially suitable for the gene from zooblast including humans (with reference to patent document 3～9).

In order to schizosaccharomyces pombe to be made the protein expression from foreign structural gene as host, it usually needs promote Enter to encode the promoter of the transcription of the foreign structural gene of the protein.As the promoter, there is schizosaccharomyces pombe itself to have Promoter in some genes, and the promoter that other biological or viruses have.

As the promoter for being utilized schizosaccharomyces pombe as in the protein expression of host, schizosaccharomyces pombe sheet Promoter in the gene that body has can enumerate alcohol dehydrogenase (adh1) gene promoter, the nmt1 bases of thiamine metabolism correlation Because of related ester of Harden Young enzyme (fbp1) gene promoter of promoter, glucose metabolism, catabolite repression correlation Invertase (inv1) gene promoter (with reference to patent document 7 or 10), heat shock protein gene promoter (with reference to patent text 11) etc. offer.Additionally the virus such as known hCMV, SV40, CaMV (constitutive expression) promoter (with reference to patent document 4,6 or 12)。

Prior art literature

Patent document

Patent document 1：International Publication No. 2012/060389

Patent document 2：Japanese Patent Laid-Open 2000-106867 publication

Patent document 3：No. 2776085 publication of Japanese Patent Laid

Patent document 4：Japanese Patent Laid-Open 07-163373 publication

Patent document 5：International Publication No. 96/23890

Patent document 6：Japanese Patent Laid-Open 10-234375 publication

Patent document 7：Japanese Patent Laid-Open 11-192094 publication

Patent document 8：Japanese Patent Laid-Open 2000-136199 publication

Patent document 9：Japanese Patent Laid-Open 2000-262284 publication

Patent document 10：International Publication No. 99/23223

Patent document 11：International Publication No. 2007/26617

Patent document 12：Japanese Patent Laid-Open 5-15380 publication

Non-patent literature

Non-patent literature 1：G.Tanaka etc., Biosci.Biotechnol.Biochem., 62 (8), 1615-1618, 1998.

Non-patent literature 2：Giga-Hama and Kumagai, eds., fission yeast Schizosaccharomyces pombe In exogenous gene expression (Foreign gene expression in fission yeast Schizosaccharomyces pombe),Springer-Verlag,(1997).

The content of the invention

Invent technical problem to be solved

In the culture of yeast, sometimes the pH of nutrient solution can switch to acidity.Especially, in the acidity of a large amount of synthesis secretions In the case of protein, the pH of the nutrient solution at the end of in most cases cultivating is changed into 2～5 acidity.Additionally, in the training of yeast Support in, in view of target protein it is productive in the case of, sometimes cultivate when optimal pH be less than 5.

Although for this purpose, for example there is flocculability in the nutrient solution of the higher pH in pH more than 5 but in the acidity of pH2～5 Under the conditions of then do not flocculate in the case of, in order that Yeast Flocculation, it is necessary to terminate the laggard pH that is about in culture and be adjusted near neutral Neutralisation treatment.

Yeast described in patent document 1 is the schizosaccharomyces pombe mutant with asexual flocculability, it is believed that be suitable as For the host of above-mentioned expression system.But, although confirm the schizosaccharomyces pombe mutant in YPD culture mediums (generally, Asexual flocculation in pH5.6～6.0), but whether also there is in acid condition enough flocculabilities to be still not clear.

Then the present invention provide it is a kind of do not need numerous and diverse separation circuit to produce, the grain wine that reclaims β-glucosyl enzym splits Grow the transformant of yeast mutants, and the manufacture method of the β-glucosyl enzym using the transformant.

Additionally, the present invention offer Novel promoter it is related, using Schizosaccharomyces yeast as host in genetic engineering In can high efficient expression the protein expression carrier from foreign gene, for making the cloning vector of the expression vector, The manufacture method of the expression vector, the transformant comprising the expression vector, the manufacture method of the transformant, and using the conversion The manufacture method of the protein of body.

Solve the technical scheme that technical problem is adopted

The transformant of the schizosaccharomyces pombe mutant of the present invention, is the increase of Gsf activity and pyruvic acid transferase Pvg1 Enzymatic activity decline or the transformant of schizosaccharomyces pombe (Schizosaccharomyces pombe) mutant for inactivating, it is special Levy and be, in chromosome or as dyeing outer-gene, the knot with the coding β-glucosyl enzym from filamentous fungi Structure gene order and promoter sequence and terminator sequence for expressing the structural gene.

Additionally, in the transformant of the schizosaccharomyces pombe mutant of the present invention, the preferred BGL1 of above-mentioned β-glucosyl enzym.

Additionally, in the transformant of the schizosaccharomyces pombe mutant of the present invention, the preferred aspergillus of above-mentioned filamentous fungi (Aspergillus) microorganism of category.

Additionally, in the transformant of the schizosaccharomyces pombe mutant of the present invention, above-mentioned β-glucosyl enzym is preferably compiled by sequence Amino acid sequence shown in number 1 is constituted, or be there occurs disappearance, taken by the amino acid for having more than 1 in the amino acid sequence The amino acid sequence of generation or addition is constituted, and the active β-glucose of the hydrolysis with catalysis β-D- glucopyranose glycosidic bonds Glycosides enzyme.

And, the manufacture method of β-glucosyl enzym of the present invention is characterised by, above-mentioned transformant is cultivated, from obtaining Thalline or nutrient solution supernatant in obtain β-glucosyl enzym.

Hereinafter, the present invention of the transformant of the structural gene sequence with above-mentioned coding β-glucosyl enzym is referred to as into the present invention First embodiment.

The cloning vector of the present invention is characterised by possessing：The startup that the hsp9 genes of Schizosaccharomyces yeast have The promoter that son or ihc1 genes have, it is positioned at the downstream of the promoter and outer for importing by the promoter control The cloning site of source structure gene, and the terminator that can be worked in Schizosaccharomyces yeast.

The promoter (hereinafter also referred to as hsp9 promoters) that hsp9 genes have preferably includes the ORF's of hsp9 genes The region of the 1～400bp of upstream of 5 ' ends, the more preferably base sequence represented by sequence number 6 or in the base sequence The base for having more than 1 there occurs replacement, disappearance or the base sequence composition added, and with promoter activity.

The promoter (hereinafter also referred to as ihc19 promoters) that ihc1 genes have preferably includes the ORF of ihc19 genes The region of the 1～501bp of upstream of the 5 ' ends of (ORFs), more preferably the base sequence represented by sequence number 9 or The base for having more than 1 in the base sequence there occurs replacement, disappearance or the base sequence composition added, and with promoter Activity.

The manufacture method of the expression vector of the present invention is characterised by, imports on the cloning site in above-mentioned cloning vector Foreign structural gene, the expression vector of the present invention is to be imported with foreign structural gene on cloning site in above-mentioned cloning vector Expression vector.

The manufacture method of the transformant of the present invention is characterised by, by above-mentioned expression vector importing Schizosaccharomyces yeast, The transformant of the present invention is the transformant containing above-mentioned expression vector.

The manufacture method of present protein is characterised by, above-mentioned transformant cultivated, from the thalline for obtaining or The protein coded by above-mentioned foreign structural gene is obtained in nutrient solution supernatant.

Hereinafter, the above-mentioned cloning vector with hsp9 promoters or ihc1 promoters, expression vector and conversion will be related to The present invention of body, and it is related to the manufacturer of the manufacture method of above-mentioned expression vector, the manufacture method of transformant and protein The present invention of method is referred to as second embodiment of the present invention.

The effect of invention

If the transformant of the schizosaccharomyces pombe mutant using the present invention, can not need numerous and diverse separation circuit In the case of production, reclaim β-glucosyl enzym.

Description of the drawings

Fig. 1 is the structure chart of expression vector pSL6AaBGL1.

Fig. 2 is the structure chart of expression vector pSL6P3AaBGL1.

Fig. 3 is the structure chart of expression vector pSL14P3AaBGL1.

Fig. 4 is the structure chart of expression vector pUC19-ura4.

Fig. 5 is the chart of the growing microorganism for representing usual strain and flocculation strain in test example 8.

Fig. 6 (a) is the chart of the concentration of glucose in the nutrient solution for represent the usual strain and flocculation strain in test example 8. Fig. 6 (b) is the chart of the concentration of alcohol in the nutrient solution for represent the usual strain and flocculation strain in test example 8.

Fig. 7 is the chart of the pNPG degrading activities of the AaBGL1 for representing usual strain and flocculation strain in test example 9.

Fig. 8 is the chart of the growing microorganism for representing usual strain and flocculation strain in test example 10.

Fig. 9 (a) is the chart of the concentration of glucose in the nutrient solution for represent the usual strain and flocculation strain in test example 10. Fig. 9 (b) is the chart of the concentration of alcohol in the nutrient solution for represent the usual strain and flocculation strain in test example 10.

Figure 10 is the photo of the precipitated form for representing usual strain and flocculation strain in test example 11.

Figure 11 is that the microscopy for representing usual strain and flocculation strain in test example 11 observes the photo of result.

Figure 12 is the chart for representing the growing microorganism at 30 DEG C and 34 DEG C in test example 12.

Figure 13 A are the charts for representing the concentration of glucose in the nutrient solution at 30 DEG C and 34 DEG C in test example 12.

Figure 13 B are the charts for representing the concentration of alcohol in the nutrient solution at 30 DEG C and 34 DEG C in test example 12.

Figure 14 is the chart of the pNPG degrading activities for representing the AaBGL1 at 30 DEG C and 34 DEG C in test example 12.

Figure 15 is the photo of the result for representing the SDS-PAGE in test example 13.

Figure 16 is the structure chart of expression vector pSL14-EGFP.

Figure 17 is the structure chart of expression vector pSL6-EGFP.

Figure 18 is that the fluorescence intensity for representing each nutrient solution in test example 14 (sets the fluorescence intensity of the nutrient solution of SL6E strains For the relative value in the case of 1) measurement result figure.

Figure 19 is the structure chart of polyclonal carrier pSL6.

Figure 20 is the structure chart of polyclonal carrier pSL12.

Figure 21 is the structure chart of polyclonal carrier pSL17.

Figure 22 is the structure chart of expression vector pSL12-EGFP.

Figure 23 is the measurement result of the rheological parameters' change with time of [fluorescence intensity/OD600] that represent each nutrient solution in test example 15 Figure.

Figure 24 is the structure chart of polyclonal carrier pSL9.

Figure 25 is the structure chart of polyclonal carrier pSL14.

Figure 26 is the structure chart of polyclonal carrier pSL14lacZ.

Specific embodiment

" foreign structural gene " refers to the gene of expression vector has coded protein, Ke Yishi in this specification Import the structural gene that the host of expression vector has in itself, it is also possible to the biological structural gene with host's xenogenesis.

" protein of foreign structural gene is derived from this specification " refer to tying from external source produced by transformant The protein of structure gene, hereinafter also referred to " exogenous proteins ".In addition, being biological with host's xenogenesis in foreign structural gene In the case of structural gene, also referred to as foreign protei matter.

First, the first embodiment of the present invention is illustrated.

[schizosaccharomyces pombe mutant]

In the present invention, as the schizosaccharomyces pombe mutant of the host of the transformant of schizosaccharomyces pombe mutant, it is After at least a portion of the gene of schizosaccharomyces pombe changes, Gsf activity increase and pyruvic acid transferase Pvg1 enzyme activity Property decline or inactivate mutant.In schizosaccharomyces pombe, if the enzymatic activity of the increase of Gsf activity and Pvg1 declines or loses It is living, even if then can also obtain the characteristic of asexual flocculation in acid condition.Hereinafter, asexual flocculation will in acid condition be carried out Property is referred to as the asexual flocculability of acid resistance.

That is, the schizosaccharomyces pombe mutant in the present invention is that of obtaining the mutant of the asexual flocculability of acid resistance.

In the present invention, " asexual flocculability " is referred to sexual the property flocculated having in itself with schizosaccharomyces pombe The property of (sexual flocculability) different flocculability.But, it is not meant to the sexual flocculability that forfeiture has in itself.Additionally, " composing type flocculability " is referred to and asexual flocculability identical property, but is refered in particular in breeding with propagation while carrying out (nothing Property ground) flocculation property.

In the present invention, " Gsf is active " refers to that schizosaccharomyces pombe (for example, shows in the pH being generally incubated under pH5～6) Asexual flocculability.The plain gene additionally, gene related to Gsf activity referred to as flocculates.

Gsf2 genes are flocculation plain gene in schizosaccharomyces pombe.The system of the gsf2 genes of schizosaccharomyces pombe is entitled SPCC1742.01。

Pvg1 is pyruvic acid transferase.The system of the pvg1 genes of the Pvg1 of coding schizosaccharomyces pombe is entitled SPAC8F11.10c。

In addition, the full base sequence of the chromosome of schizosaccharomyces pombe is in the database " GeneDB " of Sanger research institutes As " Schizosaccharomyces pombe Gene DB (http://www.genedb.org/genedb/pombe/)” It is included, discloses.The sequence data of the gene of the schizosaccharomyces pombe that this specification is recorded can be by gene name or system name Enter line retrieval acquisition in above-mentioned database.

Schizosaccharomyces pombe has the sexual flocculability of pheromones induction originally.For example, if caused in breeding Subalimentation then easily produces sexual flocculation.But, in the artificial mass propgation by groove tank culture etc., due to having Typically cultivated in the nutrient solution of enough nutrition, therefore sexual flocculation is seldom occurred.On the other hand, due in the present invention Schizosaccharomyces pombe mutant there is asexual flocculability, even if therefore cultivated in the nutrient solution with enough nutrition, Can also occur to flocculate (composing type flocculation) while propagation.

The Gsf activity of schizosaccharomyces pombe mutant for example can increase it by increasing the expression of gsf2 genes. Additionally, the enzymatic activity of the Pvg1 of schizosaccharomyces pombe can be led by making the pvg1 gene delections of coding Pvg1 or on the gene Enter to make Pvg1 enzymatic activity decline or inactivate mutation come make its decline or inactivate.

For this purpose, the schizosaccharomyces pombe mutant in the present invention can not have wild strain etc. by gene engineering method The schizosaccharomyces pombe of the asexual flocculability of acid resistance, using gene engineering method, integrates the gsf2 genes of external source simultaneously as host Make the gene delection of coding Pvg1 or import on the gene to be manufactured the enzymatic activity decline or the mutation of inactivation of Pvg1.

The expression of gsf2 genes can increase it by integrating the gsf2 genes of external source with gene engineering method.In place Again it is important that gsf2 genes are imported in main, and the gsf2 genes of importing can be of the same race with the endogenous gsf2 genes of host, The gsf2 genes of heterogeneity biological can be derived from.

Method as gsf2 genes are imported in host by gene engineering method, can use known method.As Using schizosaccharomyces pombe as host, the method that the structural gene of external source is imported wherein, for example, can use Japanese Patent Laid-Open Flat 5-15380 publications, No. 95/09914 text of International Publication No., Japanese Patent Laid-Open 10-234375 publication, Japan are specially Sharp JP 2000-262284 publication, Japanese Patent Laid-Open 2005-198612 publication, International Publication No. 2010/087344 Method described in publication etc..

Gsf2 genes are preferably imported in the chromosome of schizosaccharomyces pombe.By importing gsf2 genes in chromosome, The transformant of the maintenance excellent in stability that can obtain passing on.Further, it is also possible to import multiple gsf2 genes in chromosome.Pass through Multiple gsf2 genes are imported, the expression efficiency of gsf2 genes can be improved.In schizosaccharomyces pombe mutant, chromosome is integrated into In gsf2 genes quantity preferably 1～20, more preferably 1～8.

Method as gsf2 genes are imported in chromosome, can use known method.For example, can be with above-mentioned Japan specially Method described in sharp JP 2000-262284 publication imports multiple gsf2 genes in chromosome.Further, it is also possible to this Method imports 1 gsf2 gene in chromosome.Additionally, as described later, one or more can be imported with many places in chromosome Gsf2 genes.

Have comprising gsf2 bases as by the method in the chromosome of gsf2 channel genes schizosaccharomyces pombes, preferably using The expression cassette of cause and the carrier (hereinafter referred to as gsf2 carriers) of recombination site, the method imported by homologous recombination method.

Gsf2 carriers have the expression cassette comprising gsf2 genes and recombination site.

Expression cassette is the combination of DNA necessary in order to express Gsf2, including gsf2 genes and in schizosaccharomyces pombe The promoter for working and the terminator worked in schizosaccharomyces pombe.Furthermore, it is possible to including 5 '-untranslated region, 3 '- Any more than a kind of untranslated region.Furthermore, it is possible to including auxotroph complementary indicia.Preferred expression box is to include gsf2 Gene, promoter, terminator, 5 '-untranslated region, 3 '-untranslated region, the expression cassette of auxotroph complementary indicia.Table Can also there are multiple gsf2 genes up in box.The quantity preferably 1～8, more preferably 1～5 of the gsf2 genes in expression cassette.

As long as the promoter worked in schizosaccharomyces pombe and terminator in acid condition also can in conversion and Mutation work in vivo and maintain the expression of Gsf2.As the promoter worked in schizosaccharomyces pombe, can The promoter (the high promoter of preferred transcription initiation activity) being had in itself using schizosaccharomyces pombe or schizosaccharomyces pombe sheet The promoter (promoter from virus etc.) that body does not have.There may be promoter of more than two kinds in carrier.

As the promoter that schizosaccharomyces pombe has in itself, for example, can enumerate alcohol dehydrogenase gene promoter, thiamines The related ester of Harden Young enzyme gene promoter of the related nmt1 gene promoters of plain metabolism, glucose metabolism, decomposition generation Thank promoter (with reference to No. 99/23223 text of International Publication No.), heat shock protein gene that thing prevents the invertase gene of correlation Promoter (with reference to No. 2007/26617 text of International Publication No.) etc..As the promoter that schizosaccharomyces pombe itself does not have, Japanese Patent Laid-Open 5-15380 publication, Japanese Patent Laid-Open 7-163373 publication, Japan Patent can for example be enumerated special Open the promoter etc. from zooblast virus described in flat 10-234375 publications.In the promoter, preferred expression effect The good nmt1 gene promoters of rate and its change promoter (for example, nmt1+, nmt41), hCMV promoters, SV40 promoters.

It is also preferable to using hsp9 promoters or ihc1 promoters in second embodiment of the present invention described later.

As the terminator worked in schizosaccharomyces pombe, the terminator that can be in itself had using schizosaccharomyces pombe Or the terminator that schizosaccharomyces pombe itself does not have.There may be terminator of more than two kinds in carrier.

As terminator, for example, can enumerate Japanese Patent Laid-Open 5-15380 publication, Japanese Patent Laid-Open 7- The terminator from the mankind described in No. 163373 publications, Japanese Patent Laid-Open 10-234375 publication, preferred people's fat The terminator of cortex protein I.

The recombination site of carrier is that have to click through with the target position of the homologous recombination in the chromosome of schizosaccharomyces pombe The position of the base sequence of row homologous recombination.In addition, target site is the internal integration expression box of dyeing in schizosaccharomyces pombe Target site.Target site can be by being set to the base sequence of the recombination site of carrier to carry out the alkali of homologous recombination with the target site Basic sequence freely sets.

The homology of the base sequence of above-mentioned recombination site and the base sequence of target site is necessary for more than 70%.Additionally, From being susceptible to from the aspect of homologous recombination, the homology of the base sequence of recombination site and the base sequence of target site is preferred More than 90% is set to, more than 95% is more preferably set to.By using the carrier with such recombination site, by homologous recombination Expression cassette is incorporated into into target site.

Length (base number) preferably 20～2000bp of recombination site.If the length of recombination site is in more than 20bp, It is susceptible to homologous recombination.In addition, if the length of recombination site is in below 2000bp, then easily prevent carrier become it is long and The problem for being difficult to homologous recombination.The length of recombination site more preferably more than 100bp, further preferred more than 200bp.This Outward, the length of recombination site more preferably below 800bp, further preferred below 400bp.

Carrier can have other region of DNA domains beyond above-mentioned expression cassette and recombination site.For example can enumerate in large intestine Necessary replication initiation region, the antibiotics resistance gene (neomycin resistance gene etc.) for being referred to as " ori " of endobacillary duplication Deng.These are typically required gene in the case of using Escherichia coli carrier construction.But, as described later, preferably Above-mentioned replication initiation region is removed when as described later by the chromosome of vector integration to host.

Carrier is the carrier with cyclic DNA structure or linear DNA structure, in the cell for importing to schizosaccharomyces pombe Shi Youxuan is imported with linear DNA structure.That is, for carriers with cyclic DNA structure such as the DNAs that is usually used In the case of, it is to import in schizosaccharomyces cell after wire preferably to be cut in carrier with restriction enzyme.

In this case, the position for cutting the carrier with cyclic DNA structure is located in recombination site.Thereby, cutting The two ends of carrier partly there is recombination site respectively, by homologous recombination by the target site of whole vector integration to chromosome.

If carrier can be made into the linear DNA structure that two ends are respectively present a part for recombination site, can also With the method beyond the method cut with the carrier of cyclic DNA structure is built.

As carrier, for example can suitably using the source such as pBR322, pBR325, pUC118, pUC119, pUC18, pUC19 In colibacillary plasmid.

In this case, for necessary to the preferred duplication removed in Escherichia coli of plasmid vector during homologous recombination The referred to as replication initiation region of " ori ".Thereby, can improve above-mentioned vector integration to integration efficiency during chromosome.

Construction method to eliminating the carrier in replication initiation region is not particularly limited, and preferably uses Japanese Patent Laid-Open Method described in 2000-262284 publications.That is, preferably build in advance and inserted again on the cutting position in recombination site The precursor carrier of initiation region processed, the method for cutting off while linear DNA structure is made as previously mentioned replication initiation region.Mat This, the carrier in the replication initiation region that can easily be removed.

In addition it is also possible to the method being discussed further below：Using Japanese Patent Laid-Open 5-15380 publication, Japan Patent Unexamined Patent 7-163373 publication, No. 96/23890 text of International Publication No., Japanese Patent Laid-Open 10-234375 publication etc. Described in expression vector and its construction method, the precursor carrier with expression cassette and recombination site is built, further with normal The genetic engineering gimmick of rule removes replication initiation region from the precursor carrier, obtains the carrier for homologous recombination.

The target site of integration vector can only exist 1 in the chromosome of schizosaccharomyces pombe, it is also possible to exist 2 with On.Target site is present in the case of more than 2, and more than 2 carriers can be integrated in the chromosome of schizosaccharomyces pombe.Additionally, In the case that gsf2 genes in expression cassette are multiple, multiple gsf2 genes can be integrated on 1 target site.And, also may be used With the carrier of more than two kinds that there is the recombination site for corresponding respectively to each target site used in target site of more than two kinds, integrate Expression cassette.

In the case of integration expression box on 1 target site, for example, can use Japanese Patent Laid-Open 2000-262284 public affairs Target site described in report.The carrier of more than two kinds with different integration sites can be used, on different target sites respectively Integration vector.But, in the case of more than 2 position integration vectors of chromosome, the method is numerous and diverse.

If can using the base sequence part for being present in multiple positions in chromosome and being substantially identical to each other as Target site, distinguishes integration vector on the target site of the plurality of position, then can use a kind of carrier at 2 of chromosome with upper Put integration vector.The base sequence being substantially identical to each other refers to the homology of base sequence more than 90%.It is preferred that target site Between homology more than 95%.Additionally, the length of the base sequence being substantially identical to each other is the weight for including above-mentioned carrier The length in group site, preferred more than 1000bp.Compared with the situation of multiple gsf2 genes is integrated on 1 target site, even if The integration quantity of gsf2 genes is identical, is being present on the target site of multiple positions in the case of diffused integration gsf2 genes, turns Change the possibility that gsf2 genes all come off quickly from chromosome when body is bred to diminish, maintenance of the transformant in passing on is steady Qualitative raising.

As the target site for being present in multiple positions in chromosome, preferred transposon gene Tf2.Tf2 is split in grain wine Grow the transposon gene that 13 altogether are respectively present on 3 (monoploid) chromosomes of yeast, length (base number) about 4900bp, Known these intergenic base sequence homologys are 99.7% (with reference to following documents).

Nathan J.Bowen et al,“Retrotran sposons and Their Recognition of pol II Promoters：A Comprehensive Survey of the Transposable Elemen ts From the Complete Genome Sequen ce of Schizosaccharomyces pombe”,G enome Res.2003 13： 1984-1997

Can only by vector integration to 1 be present in chromosome in the Tf2 of 13 positions.In this case, By integrating the carrier with more than 2 gsf2 genes, the transformant with more than 2 gsf2 genes can be obtained.Additionally, passing through Integration vector on Tf2 more than 2, can obtain the transformant with more than 2 gsf2 genes.In this case, by whole The carrier with more than 2 gsf2 genes is closed, the transformant with more gsf2 genes can be obtained.If complete on 13 Tf2 Portion's integration vector, then the burden that the existence to transformant or propagation are caused may be excessive.It is preferred that 8 in 13 Tf2 with Go up integration vector down, more preferably below 5 on integration vector.

Being used as host in the case where the schizosaccharomyces pombe mutant in the present invention is manufactured by gene engineering method is made Schizosaccharomyces pombe, preferably with for selecting the mark of transformant.For example, it is preferable to using certain gene delection in growth The middle host for needing specific nutrition composition.By the gene (auxotroph complementary indicia) for integrating the disappearance in the carrier, turn The auxotrophy for changing host in body disappears.

By the host and the auxotrophic difference of transformant, both can be distinguished, obtain transformant.

For example, the uracil-deficient type grain wine fragmentation ferment for orotidylic decarboxylase gene (ura4 genes) being lacked or being inactivated Mother is phonetic by selection urine after being converted by the carrier with ura4 genes (auxotroph complementary indicia) as host The cell that pyridine deficiency disappears, can obtain incorporating the transformant of carrier.Cause auxotrophic base due to lacking in host As long as the selection because can be used for transformant, is not limited to ura4 genes, or isopropylmalate dehydrogenase gene (leu1 genes) etc..

Generally, carry out after homologous recombination, select the transformant for obtaining.Method alternatively, for example, can enumerate with shown below The method for going out.Using the culture medium of transformant can be selected to be screened by above-mentioned nutrient defect type mark, from the bacterium for obtaining Multiple bacterium colonies are selected in falling.Then, these are carried out respectively after Liquid Culture, investigates the gsf2 bases of respective thalline per unit The expression of cause, the mutant for selecting the expression more.Additionally, by carrying out to the mutant that these are selected based on pulse The genome analysis of field gel electrophoresis method, can investigate out the carrier number and expression cassette number being incorporated in chromosome.

The carrier number integrated in chromosome can pass through the adjustment that adjustment integration condition etc. carries out to a certain degree, but think basis The size (base number) of carrier and structure, integration efficiency and integration quantity also can change.

By with gene engineering method make pvg1 gene defects or in pvg1 genes import decline the enzymatic activity of Pvg1 Or the mutation of inactivation, the enzymatic activity of the Pvg1 of the schizosaccharomyces pombe as host can be made to decline or inactivate.In order to can be certain Inactivate the enzymatic activity of Pvg1, preferably make pvg1 genes itself defect from chromosome.

The available known method of disappearance or inactivation of pvg1 genes is carried out.For example, can be by using Latour methods (Nucreic Acids Res magazines, 2006, volume 34, e11 page, described in No. 2007/063919 text of International Publication No. etc.) Make pvg1 gene delections.

Additionally, a part for the base sequence by making pvg1 genes occurs disappearance, insertion, replacement, addition, this can be also made Pvg1 genes are inactivated.Due to the disappearance of the gene, insertion, replacement, addition and caused mutation can only occur appointing in these It is a kind of, it is also possible to which that two or more occurs.

The method of above-mentioned mutation is imported in a part for pvg1 genes can use known method.Employing can for example be enumerated The mutation partition method (molecular genetics in yeast experimental method 1996, learns publishing centre) of mutagens, dashing forward at random using PCR Political reform (PCR method application (PCR Methods Appl.), 1992, volume 2, p.28-33.) etc..

The common grain wine that schizosaccharomyces pombe mutant in the present invention also for example never can have asexual flocculability splits Grow yeast to obtain by induced mutations means.That is, can make as follows：By the schizosaccharomyces pombe to there is no asexual flocculability Mutagenic treatment is carried out, the enzyme activity of the increase of Gsf activity and Pvg1 compared with wild strain is selected in the schizosaccharomyces pombe from after process Property the bacterium that declines or inactivate, further select the increase of Gsf activity or decline of enzymatic activity etc. of Pvg1 to be aobvious from the bacterium for selecting Property mutation bacterium.

The mutagenic treatment of schizosaccharomyces pombe also be able to can be shone using the mutation evocating substance such as EMS (ethylmethane sulfonate) Penetrate the light of the short wavelengths such as ultraviolet.Additionally, the schizosaccharomyces pombe screening from after mutagenic treatment has the asexual flocculability of acid resistance Bacterium can carry out in the presence of the cations such as calcium ion.

The Gsf activity of schizosaccharomyces pombe mutant can be evaluated using settling velocity as index.For this purpose, will lure Schizosaccharomyces pombe after change is processed is cultivated on solid medium, the bacterium colony of formation is put into into the feelings in appropriate solvent Under condition, in the case where the bacterium colony than wild strain significantly precipitates (settling velocity is fast) earlier, the bacterium for forming the bacterium colony can be evaluated It is the Gsf activity increase compared with wild strain.The bacterium colony of the bacterium colony of wild strain and the bacterium after mutagenic treatment almost can simultaneously be put into Solvent, compares the speed of precipitation, the settling velocity of the wild strain under specified conditions also can be in advance determined, by by the result for obtaining The settling velocity of the bacterium colony of the bacterium after fixed threshold value and mutagenic treatment is compared.As long as the solvent of the precipitation test for bacterium colony Solution that yeast can survive, there is no particular limitation, preferably comprise selected from calcium ion, lithium ion, manganese ion, copper from The buffer solution of the cation of more than a kind of sub and zinc ion.For example, in the case where the concentration for being dried thalline is 3.6g/L, Settling velocity in the lactic acid buffer (80mM lactic acid, 100mM calcium chloride, pH6.0) of calcium ions is the bacterium of more than 1.0m/h Can be as the screening mutant of Gsf activity increases out.

For example, the asexual flocculation strain of acid resistance can be obtained by following operation.First-selection, to schizosaccharomyces pombe EMS is used After Mutation induction, culture is isolated.Afterwards, the thalline for removal supernatant being reclaimed is according to the concentration for being dried thalline for 3.6g/L's Condition is suspended in lactic acid-sodium hydrate buffer solution (80mM lactic acid, 100mM calcium chloride, pH2.0), determines settling velocity, will be heavy Bacterial strain of the shallow lake speed more than 2.0m/h is selected good strains in the field for seed as the asexual flocculation of acid resistance and is selected.

The mutant of Gsf activity increases also can be screened the expression of gsf2 genes as index.Gsf2 genes Expression can pass through RT-PCR, the measure being usually used in gene expression analysis using the RNA traces etc. of marked probe Method is measured.

If the enzymatic activity of Pvg1 declines or inactivates, what the amount of the pyruvic acid of cell surface layer was decreased significantly inclines To.For this purpose, can comment the enzymatic activity of the Pvg1 of schizosaccharomyces pombe using the amount of cell surface layer pyruvic acid as index Valency.That is, in schizosaccharomyces pombe that can be from after mutagenic treatment using the bacterium of deletion cells top layer pyruvic acid as Pvg1 enzymatic activity The screening mutant for declining or inactivating is out.

The deletion mutant of cell surface layer pyruvic acid refers to method (the The Journal of of Andreishcheva etc. biological chemistry(《Biochemistry periodical》) 2004 on Augusts 20,；279(34)：35644-55) made. First-selection, to schizosaccharomyces pombe using after EMS Mutation inductions, is used 48 hours of appropriate fluid nutrient medium culture.Afterwards, The thalline for adsorbing is removed from culture using positively charged Q- Ago-Gels, reclaims the thalline remained in supernatant.It is logical Cross and be repeated after sorting for several times using the Q- Ago-Gels, the nutrient solution supernatant for obtaining is coated on plate carries out separation training Support, the deletion mutant of cell surface layer pyruvic acid is thus obtained.

The enzymatic activity of Pvg1 declines or the mutant of inactivation also can be by the enzyme of the Pvg1 of each bacterium after measure mutagenic treatment Activity is screened.The enzymatic activity of the Pvg1 of schizosaccharomyces pombe can be by using assay method of marked substrate etc. at it Commonly used approach is measured in the enzyme assay of its transferase.

Schizosaccharomyces pombe mutant in the present invention can also be manufactured gene engineering method and mutagenic treatment combination. For example, can make Pvg1's using gene engineering method to the mutant that the expression of gsf2 genes is increased by mutagenic treatment Enzymatic activity declines or inactivates, it is also possible to which the enzymatic activity to being made Pvg1 by mutagenic treatment is declined or the mutant of inactivation utilizes gene Engineering method increases the expression of gsf2 genes.The enzymatic activity that Pvg1 is made by gene engineering method can also be declined or mistake Mutant living carries out mutagenic treatment, screens the mutant that the expression of gsf2 genes is increased.

Schizosaccharomyces pombe mutant in the present invention is can maintain can be at it in the range of the asexual flocculability of acid resistance There is mutation on its gene, can be with chromosome or dyeing is imported with vitro the structural gene of external source.

The asexual flocculability of acid resistance that schizosaccharomyces pombe mutant in the present invention possesses is not by the acid in nutrient solution Species impact.That is, no matter make nutrient solution pH be reduced to 2～5 acid be lactic acid, citric acid, acetic acid, butanedioic acid, fumaric acid, The organic acids such as malic acid, or the inorganic acid such as hydrochloric acid and sulfuric acid, the schizosaccharomyces pombe mutant all carries out asexual flocculation.

The intensity of the asexual flocculability that the schizosaccharomyces pombe mutant in the present invention possesses can for example use settling velocity As index.For the settling velocity of yeast, for example, can be suspended by the yeast thalline that will be divided in the transparent vessels such as test tube Stand after process, after starting precipitation, will be from liquid surface to solid liquid interface (the yeast thalline of precipitation and the interface of supernatant) Distance is obtained divided by the elapsed time precipitated after starting.

Schizosaccharomyces pombe mutant in the present invention have the lactic acid buffer containing calcium ion (80mM lactic acid, 100mM calcium chloride, pH2.0) in asexual flocculation property.Schizosaccharomyces pombe mutant in the present invention this contain calcium from Settling velocity in the lactic acid buffer of son, in the case where the concentration for being dried thalline is 3.6g/L, preferred more than 2.0m/h, more It is preferred that more than 4.0m/h, further preferred more than 6.0m/h.

Additionally, the schizosaccharomyces pombe mutant in the present invention has, and in the lactic acid buffer containing calcium ion, (80mM is newborn Acid, 100mM calcium chloride, pH4.0) in asexual flocculation property.Schizosaccharomyces pombe mutant in the present invention contains at this Settling velocity in the lactic acid buffer of calcium ion, be dried thalline concentration be 3.6g/L in the case of, preferred 2.0m/h with On, more preferably more than 4.0m/h, further preferred more than 8.0m/h.

Settling velocity in by making pH4.0 be more than 8.0m/h, with pH be 4 and lower pH in compared with, can show Go out sufficient flocculability.

The asexual flocculability of acid resistance that schizosaccharomyces pombe mutant in the present invention possesses may also rely on selected from calcium More than a kind cation of ion, lithium ion, manganese ion, copper ion and zinc ion.Depend in the asexual flocculability of acid resistance In the case of calcium ion etc., the schizosaccharomyces pombe mutant can be suppressed by adding the chelating agents such as EDTA in the medium Flocculation.

The asexual flocculability of acid resistance that schizosaccharomyces pombe mutant in the present invention possesses can also be using half Lactose is come the property that suppresses.In the case where the asexual flocculability of acid resistance is suppressed using galactolipin, by pressing in nutrient solution According to the condition addition galactolipin for making ultimate density be more than 5mM, the flocculation of the schizosaccharomyces pombe mutant can be suppressed.

Schizosaccharomyces pombe mutant in the present invention (for example, shows strong asexual wadding acid under the conditions of pH2～5) Solidifying property.For this purpose, the schizosaccharomyces pombe mutant is particularly suitable as the host of synthetic acidic protein expression system. In addition, it is contemplated that the productivity of target protein, even if its result is the optimal pH in culture being less than in the case of 5, also fits Cooperate the host for expression system.

, as host, β-glucosyl enzym is manufactured in a large number, can make in order to using the schizosaccharomyces pombe mutant in the present invention The transformant of the schizosaccharomyces pombe mutant of the present invention described later, in the feelings for being cultivated the transformant with groove tank culture etc. Under condition, even if in the case that the pH of nutrient solution is 2～5 at the end of culture, the solid-liquid such as can not also be centrifuged, filter and dividing Just make thalline flocculate from process or neutralization reaction etc., can thereby be easily separated thalline and nutrient solution.In addition, the grain in the present invention Wine fission yeast mutant not only in acid condition, (for example, also can be asexual under the conditions of pH5～10) in faintly acid～alkalescence Flocculation.

[transformant of schizosaccharomyces pombe mutant]

The present invention schizosaccharomyces pombe mutant transformant using above-mentioned schizosaccharomyces pombe mutant as host, There is expression cassette in chromosome or as dyeing outer-gene, the expression cassette includes the coding β-glucose from filamentous fungi The structural gene sequence of glycosides enzyme and promoter sequence and terminator sequence for expressing the structural gene.Have in chromosome There is above-mentioned expression cassette to refer to more than at expression cassette is incorporated in the chromosome of Schizosaccharomyces yeast 1, as the external base of dyeing Have in the cell the plasmid containing expression cassette because having above-mentioned expression cassette to refer to.In terms of the Secondary Culture of transformant is easy Set out, there is above-mentioned expression cassette preferably in chromosome.

In addition, expression cassette is identical with described in [schizosaccharomyces pombe mutant], it is to must to express β-glucosyl enzym The combination of the DNA for needing, including β-glucosyl enzym structural gene and the promoter that works in Schizosaccharomyces yeast and termination Son.

Additionally, the β-glucosyl enzym from the cell exocrine to Schizosaccharomyces yeast increase, the recovery of β-glucosyl enzym, Purifying readily from the point of view of aspect, preferably has in 5 ' end sides of β-glucosyl enzym structural gene and is risen in Schizosaccharomyces yeast The base sequence (structural gene of secretion signal) of the encoding secretion signals sequence of effect.The 5 ' of β-glucosyl enzym structural gene are last Side is referred in 5 ' end side upstreams of β-glucosyl enzym structural gene, adjacent with 5 ' ends of β-glucosyl enzym structural gene Position.Furthermore, it is possible to the base sequence of active, coding N-terminal side the several amino acid for not affecting β-glucosyl enzym is removed, The gene of coded signal sequence is imported on the position.

It is that as long as promoter and terminator can in vivo work in the schizosaccharomyces pombe mutation as host, can express From the promoter and terminator of the β-glucosyl enzym of filamentous fungi.Act as in vivo as being mutated in schizosaccharomyces pombe Promoter, can enumerate and identical promoter described in [schizosaccharomyces pombe mutant].

(β-glucosyl enzym)

β-glucosyl enzym (EC.3.2.1.21) is used as the hydrolysis of specific catalytic β-D- glucopyranose glycosidic bonds The general name of enzyme.Be particularly due to cellobiose can be decomposed into into glucose and be referred to as cellobiase, be distributed widely in bacterium, In filamentous fungi, plant and animal.In various species, the gene of multiple coding β-glucosyl enzyms is usually present, for example, Presence (the Soy of bgl1～bgl7 is reported in the aspergillus oryzae (Aspergillus oryzae) of a kind as filamentous fungi protein Research(《Soybean protein is studied》), Japan 12,78-83,2009, Japanese Patent Laid-Open 2008-086310 Publication).Wherein, from it is active it is high in terms of from the point of view of, the bgl1 of optimized encoding BGL1.

The structural gene of the β-glucosyl enzym that the transformant of the schizosaccharomyces pombe mutant of the present invention has is derived from Filamentous fungi.

Filamentous fungi referred in mushroom, the eukaryotic cell microorganism being made up of the tubular fossils for being referred to as mycelia.As Filamentous fungi, for example, can enumerate aspergillus (Aspergillus), trichoderma (Trichoderma) category, Fusarium (Fusarium), Penicillium (Penicillium) and Acremonium (Acremonium) etc..β-glucosyl enzym in the present invention Structural gene can be derived from any one filamentous fungi β-glucosyl enzym structural gene, as long as the filamentous fungi be produce The filamentous fungi of raw β-glucosyl enzym, in terms of enzymatic activity height etc., is preferably derived from the filamentous fungi of aspergillus β-glucosyl enzym.As the filamentous fungi of aspergillus, for example, can enumerate aspergillus nidulans (Aspergillus nidulans), meter Qu Mould (Aspergillus oryzae), microorganism Aspergillus aculeatus (Aspergillus aculeatus), aspergillus niger (Aspergillus Niger), powdery aspergillus (Aspergillus pulverulentus).From decomposition and crystallization cellulose ability height, generate monose energy From the point of view of the excellent aspect of power, the coding β-glucosyl enzym of microorganism Aspergillus aculeatus (Aspergillus aculeatus) is preferably derived from Gene, more preferably from the coding BGL1 (hereinafter also referred to as AaBGL1) of microorganism Aspergillus aculeatus (Aspergillus aculeatus) Gene.

According to the youth's thesis for the doctorate of slope this gift one (with regard to the cellulose enzyme of Aspergillus aculeatus No.F-50 Research, Osaka Prefecture University, 1984), from the purifying of Aspergillus aculeatus wild type AaBGL1 point Son amount about 133KDa, optimal pH is 4.0, and stable pH range is 3～7 (25 DEG C, 24 hours).

The amino acid sequence sequence number 1 of AaBGL1 is represented.The gene order that β-glucosyl enzym is encoded in the present invention is excellent Gene order that amino acid sequence of the choosing represented by sequence number 1 is constituted, coding β-glucosyl enzym.In addition it is also possible to be There are 1～tens, the ammonia of preferably 1～several, further preferred 1～9 in amino acid sequence represented by sequence number 1 Base acid there occurs that the amino acid sequence of disappearance, replacement or addition is constituted, coding has and be catalyzed β-D- glucopyranose glycosidic bonds The gene order of the active β-glucosyl enzym of hydrolysis.

Even if there is the amino of 1～tens in the β-glucosyl enzym that the amino acid sequence represented by sequence number 1 is constituted Acid disappearance, replace or add, it may have catalysis β-D- glucopyranose glycosidic bonds hydrolysis activity.

In addition, the gene of the above-mentioned coding β-glucosyl enzym from filamentous fungi can be used directly, but in order that split The expression increase in Saccharomyces cerevisiae is grown, preferably said gene sequence the Gao Biaodaji in Schizosaccharomyces yeast is changed to into The high codon of frequency because used in.

In the present invention, as expressing the carrier (hereinafter also referred to as bgl carriers) of β-glucosyl enzym, can enumerate with it is upper The gsf2 carrier identical carriers for expressing Gsf2 stated.

And, bgl carriers preferably have the secretion signal gene worked in vivo in schizosaccharomyces pombe mutation.Secretion letter The position of number gene for β-glucosyl enzym structural gene 5 ' end sides.In the secretion that schizosaccharomyces pombe mutation is worked in vivo Signal gene is the gene of the amino acid sequence of the function that coding is secreted into outside host cell with the exogenous proteins for making expression. The exogenous proteins that secretion signal is with the addition of on N-terminal are expressed by the foreign structural gene for combining secretion signal gene. Secretion signal in endoplasmic reticulum or golgiosome in host etc. on the exogenous proteins is cut, and afterwards, eliminates secretion The exogenous proteins of signal are secreted into extracellular.Secretion signal gene (and secretion signal) is had in schizosaccharomyces pombe Mutation is worked in vivo.As the secretion signal gene worked in vivo in schizosaccharomyces pombe mutation, it is possible to use for example remember It is loaded in the gene of International Publication No. 1996/23890.

In the present invention, by 5 ' end sides of the structural gene in β-glucosyl enzym the structure base of the secretion signal is imported Cause, can express the β-glucosyl enzym that above-mentioned secretion signal is with the addition of on N-terminal, make β-glucosyl enzym be secreted into Schizosaccharomyces Outside the thalline of yeast.As the secretion signal worked in Schizosaccharomyces yeast, particularly preferred International Publication No. 1996/ P3 signals described in No. 23890.

Using above-mentioned bgl carriers, the schizosaccharomyces pombe mutant as host is converted.To the schizosaccharomyces pombe mutation Importing the structural gene of β-glucosyl enzym in vivo can be carried out with gsf2 gene identical modes are imported.Additionally, the choosing of transformant Selection method is also identical.

The nutrient solution of the transformant of the schizosaccharomyces pombe mutant of the present invention can use known Yeast Cultivation culture medium, As long as containing the assimilable carbon source of Schizosaccharomyces yeast, nitrogen source, inorganic salts etc., can efficiency carry out fission yeast well The culture of category yeast.As nutrient solution, natural medium can have both been used, it is also possible to use synthetic media.

As carbon source, for example, can enumerate the sugar such as glucose, fructose, sucrose.

As nitrogen source, for example, can enumerate ammonium salt, peptone, the junket egg of the inorganic acids such as ammonia, ammonium chloride, ammonium acetate or inorganic acid Casamino acid etc..

As inorganic salts, for example, can enumerate magnesium phosphate, magnesium sulfate, sodium chloride.

Culture can adopt known rhodozyma culture method, for example, can be carried out by shaken cultivation, stir culture etc..

In addition, cultivation temperature is preferably 23-37 DEG C.Incubation time can be determined suitably.

Additionally, culture can be batch culture (batch cultures) or fed-batch culture (fed-batch cultures), also may be used Being continuous culture.

As the transformant of the schizosaccharomyces pombe mutant of the present invention, have what is combined with secretion signal gene using In the case of the transformant of β-glucosyl enzym structural gene, β-glucosyl enzym is secreted in nutrient solution.Then, in order to make in a large number β-glucosyl enzym is made and in the case that the transformant is cultivated with groove tank culture etc., even if the nutrient solution at the end of culture In the case that pH is 2～5, also due to the transformant has the asexual flocculability of acid resistance, and can not be centrifuged, filter Separation of solid and liquid process or neutralization reaction etc. just make thalline flocculate, and can thereby be easily separated thalline and nutrient solution.

Additionally, as the present invention schizosaccharomyces pombe mutant transformant, using have not with secretion signal base Because combine β-glucosyl enzym structural gene transformant in the case of, as the separation method of β-glucosyl enzym, can using public affairs The method for protein isolation known.

For example, the thalline of precipitation can be separated from nutrient solution after incubation, destroys thalline, obtained containing β-glucose The cell pulverization liquid of glycosides enzyme, saltouts, the known albumen such as post purifying, chromatography, immunoprecipitation from used in the cell pulverization liquid Matter separation method obtains β-glucosyl enzym.

Then, second embodiment of the present invention is illustrated.

[cloning vector]

The cloning vector of second embodiment of the present invention be in order to express exogenous proteins and in Schizosaccharomyces yeast The cloning vector for making expression vector of middle importing, is characterized in that, the promoter for regulating and controlling the expression of exogenous proteins is to split Grow the hsp9 promoters or ihc1 promoters of Saccharomyces cerevisiae.In addition, following also by the clone of second embodiment of the present invention Carrier is referred to as the cloning vector of the present invention.

The hsp9 genes of Schizosaccharomyces yeast are the heat shock proteins that coding has as Schizosaccharomyces yeast A kind of gene of the Hsp9 protein of (heatshock protein, hsp).The geneseq database of schizosaccharomyces pombe (S.pombe GeneDB；http://www.genedb.org/genedb/pombe/) in log in hsp9 genes system name For SPAP8A3.04c.

Heat shock protein refers to, cell or individuality are by drastically higher than the temperature change (warm of 5～10 DEG C of ordinary temperatures Shock) when its synthesis be induced, suppressed by molecular chaperones effect protein thermal denaturation and flocculate protein general name. The synthesis in vivo of the biology of heat shock protein in addition to heat shock, also can by various chemical substances, such as electron transport chain Inhibitor, transition metal, SH reagents, ethanol etc. are induced.

Therefore, in the transformant for having imported the expression vector with hsp9 promoters, with Hsp9 protein expression phases Together, the expression of exogenous proteins can be regulated and controled by the stimulation of heat shock or various chemical substances.

Expression efficiency of the hsp9 promoters in Schizosaccharomyces yeast is very high.For this purpose, by using the promoter, can The expression vector that can produce unprecedented substantial amounts of exogenous proteins is made by the transformant of Schizosaccharomyces yeast.

Hsp9 promoters can be the promoter of the hsp9 genes that Schizosaccharomyces yeast has, and can derive from arbitrary Kind of Schizosaccharomyces yeast, but due to more general and hsp9 promoters that preferably use schizosaccharomyces pombe.Grain wine fragmentation ferment Female hsp9 promoters are included in the 1～400bp of upstream of the 5 ' ends of the ORF of hsp9 genes (A of initiation codon ATG) Region (canonical sequence numbering 6).

As the Schizosaccharomyces yeast beyond the schizosaccharomyces pombe with hsp9 promoters, Japanese fragmentation ferment can be enumerated Female (Schizosaccharomyces japonicus), eight spore fission yeast (Schizosaccharomyces Octosporus) etc..Additionally, for the hsp9 promoters of cloning vector, the expression that can be made by the cloning vector with importing The Schizosaccharomyces yeast of carrier is same species source, or different plant species source.

(hsp9 of wild type is opened the promoter that hsp9 promoters have in itself except the Schizosaccharomyces yeast with wild type Mover) beyond identical base sequence, or by disappearance in the base sequence, replace or add more than 1, preferably 1 ～tens, more preferably 1～more than ten, further preferred 1～9, the base sequence structure of still more preferably 1～several bases Into and identical with the hsp9 promoters of wild type the region with promoter activity.

Additionally, the hsp9 promoters that the cloning vector of the present invention is used can also be by the hsp9 promoters with wild type The homology of identical base sequence is more than 80%, preferably more than 85%, more preferably more than 90%, further preferred 95% with On base sequence constitute and identical with the hsp9 promoters of wild type the region with promoter activity.

The hsp9 promoters of schizosaccharomyces pombe be included in the ORF of hsp9 genes 5 ' ends (initiation codon ATG A the region in 1～400bp of upstream).The base sequence in the region is shown in sequence number 6.That is, as the clone of the present invention Carrier, is preferably provided with the region that the base sequence represented by sequence number 6 is constituted.Additionally, following sequences are also suitable for this The hsp9 promoters that the cloning vector of invention is used：Disappearance, replacement or addition in base sequence represented by sequence number 6 More than 1, preferably 1～tens, more preferably 1～more than ten, further preferred 1～9, still more preferably 1～several alkali The base sequence of base, or with the homology of the base sequence represented by sequence number 6 be more than 80%, preferably more than 85%, more It is preferred that more than 90%, further preferred more than 95% base sequence is constituted, and identical with the hsp9 promoters of wild type have There is the region of promoter activity.

Ihc1 genes are the genes of the protein Ihc1 of coding molecule amount 15400.Ihc1 genes are extensively retained in fragmentation In Mycophyta headed by Saccharomyces cerevisiae.

Ihc1 protein propagation start when etc. cell density it is low in the state of be suppressed expression, in the high shape of cell density It is induced expression under state.The Ihc1 protein expressions are regulated and controled by the promoter of ihc1 genes.Therefore, ihc1 promoters Can propagation start when etc. cell density it is low in the state of suppress express induction, cell density height in the state of consumingly lure Lead expression.For this purpose, by using the promoter, can make in the transformant of Schizosaccharomyces yeast can be according to cell density To adjust the expression vector of the expression of exogenous proteins.

As long as the promoter of the ihc1 genes that ihc1 promoter Schizosaccharomyces yeast has, can derive from Any one Schizosaccharomyces yeast, but due to ihc1 promoters that are more general and preferably using schizosaccharomyces pombe.

The ihc1 genes of schizosaccharomyces pombe are known, and in the geneseq database of schizosaccharomyces pombe, (grain wine splits Grow yeast GeneDB；http://www.genedb.org/genedb/pombe/) in log in ihc1 genes system it is entitled SPAC22G7.11c, the ihc1 promoters are included in the upper of the 5 ' ends (A of initiation codon ATG) of the ORF of ihc1 genes Region (canonical sequence numbering 9) in 1～501bp of trip.

Ihc1 promoters used in the cloning vector of the present invention can be the ihc1 bases that Schizosaccharomyces yeast has The promoter of cause, it is also possible to from any one Schizosaccharomyces yeast.As Schizosaccharomyces yeast, grain wine fragmentation can be enumerated Yeast, Japanese fission yeast, eight spore fission yeasts etc..Additionally, for the ihc1 promoters of cloning vector, can be with importing by this The Schizosaccharomyces yeast of the expression vector that cloning vector makes is same species source, or different plant species source. In the present invention, due to ihc1 promoters that are more general and preferably using schizosaccharomyces pombe.

The present invention cloning vector used in ihc1 promoters except with the Schizosaccharomyces yeast of wild type itself Lack beyond promoter (the ihc1 promoters of wild type) the identical base sequence having, or by the base sequence Lose, replace or add more than 1, preferably 1～tens, more preferably 1～more than ten, further preferred 1～9, more enter one The base sequence of preferably 1～several bases of step is constituted and identical with the ihc1 promoters of wild type with promoter activity Region.

Additionally, the ihc1 promoters that the cloning vector of the present invention is used can also be by the ihc1 promoters with wild type The homology of identical base sequence is more than 80%, preferably more than 85%, more preferably more than 90%, further preferred 95% with On base sequence constitute and identical with the ihc1 promoters of wild type the region with promoter activity.

The ihc1 promoters of schizosaccharomyces pombe be included in the ORF of ihc1 genes 5 ' ends (initiation codon ATG A the region (sequence number 9) in 1～501bp of upstream).The base sequence in the region is shown in sequence number 9.That is, as this The cloning vector of invention, is preferably provided with the region that the base sequence represented by sequence number 9 is constituted.Additionally, following sequences It is suitable as the ihc1 promoters that the cloning vector of the present invention is used：Lack in base sequence represented by sequence number 9, Replace or add more than 1, preferably 1～tens, more preferably 1～more than ten, further preferred 1～9, still more preferably The base sequence of 1～several bases, or with the homology of the base sequence represented by sequence number 9 be more than 80%, preferably More than 85%, more preferably more than 90%, further preferred more than 95% base sequence is constituted, and is started with the ihc1 of wild type Son has in the same manner the region of promoter activity.

The cloning vector of the present invention outside any one of above-mentioned hsp9 promoters and ihc1 promoters, also with being located at The downstream of the promoter and the cloning site for importing foreign structural gene by promoter control and can be in fragmentation The terminator worked in Saccharomyces cerevisiae.

The cloning site that cloning vector possesses be in cloning vector only this present on cloning site, there is restriction The region of enzyme recognition site.The cloning site that the cloning vector of the present invention possesses can have by the limit of a kind of restriction enzyme identification Enzyme recognition site processed, it is possible to have the MCS of the restriction enzyme recognition site that can be recognized by two or more restriction enzyme.Make For the MCS, the MCS that known cloning vector possesses is can be used directly, furthermore, it is possible to will be known many Cloning site carries out being used after suitably changing.In addition, the cloning vector of the present invention can be in cloning site downstream side region Or the downstream of the cloning site possesses terminator codon.

As the terminator worked in Schizosaccharomyces yeast, the end that can be in itself had using Schizosaccharomyces yeast The terminator that only son or Schizosaccharomyces yeast itself do not have.In addition, there may be two or more terminators in carrier. As the terminator that Schizosaccharomyces yeast has in itself, for example, can enumerate the terminator of the inv1 genes of Schizosaccharomyces yeast Deng.Additionally, the terminator not having as Schizosaccharomyces yeast itself, such as can enumerate described in patent document 2,4 or 10 The terminator from the mankind etc., the terminator of preferred people's lipocortin I.

Preferably include 5 '-untranslated in the upstream of the downstream of above-mentioned promoter, cloning site in the cloning vector of the present invention Region, furthermore it is preferred that including 3 '-untranslated region in the downstream of cloning site.Additionally, the cloning vector of the present invention preferably has The mark of the expression vector of foreign structural gene has been imported on cloning site for identification.As the mark, for example, can enumerate Drug resistance gene that ampicillin resistance gene etc. can work in Escherichia coli etc..

And, the cloning vector of the present invention preferably has the mark for selecting transformant.As the mark, for example can example Lift auxotroph complementary indicia, the isopropylmalate dehydrogenase genes (leu1 genes) such as ura4 genes etc..

The region of composition expression cassette when the cloning vector of the present invention on the cloning site except importing foreign structural gene Outside, there can also be region of DNA domain necessary to the manufacture of transformant.For example, in the case where expression cassette is imported into chromosome It is preferred that having recombination site.Dye to directly import the expression cassette with foreign structural gene (being not limited to gsf2 genes) Body, will can be used using in the first embodiment of the invention described above in the case of the expression cassette importing chromosome of gsf2 carriers Recombination site.In the case of the expression cassette of the foreign structural gene beyond with gsf2 genes, the expression cassette is imported into dye The method of colour solid also can be used directly the gene engineering method described in the first embodiment of the present invention.

The expression cassette of the expression vector made in the cloning vector made by the present invention is in host cell as chromosome In the case of the transformant that alia gene keeps, the cloning vector of the present invention is preferably comprised for carrying out in Schizosaccharomyces yeast The sequence of duplication, i.e. autonomously replicating sequence (Autonomously Replicating Sequence：ARS).In addition, by table In the case of being incorporated in chromosome up to box, after preferably is deleted from expression vector ARS host is imported.

The cloning vector of the present invention can be by from order to make the expression vector for expressing exogenous proteins in host In the known cloning vector for being used, replace what the known cloning vector possessed to open with hsp9 promoters or ihc1 promoters Sub-area is being made.For example, can be by replacing above-mentioned Japanese Patent Laid-Open with hsp9 promoters or ihc1 promoters 7-163373 publications, Japanese Patent Laid-Open 10-234375 publication, Japanese Patent Laid-Open 11-192094 publication, day The promoter site of the polyclonal carrier described in this patent JP 2000-136199 publication etc. is being made.

As the concrete operation method of the cloning vector for being used to build the present invention, known method can be used.For example, can make With document [J.Sambrook etc., " Molecular Cloning 2nded (《The molecular cloning second edition》).",Cold Spring Harbor Laboratory Press (CSH Press) (1989)] described in method of operating.In addition, also may be used Built with the enzymatic amplification method or chemical synthesis of PCR-based.

[expression vector and its manufacture method]

The expression vector of second embodiment of the present invention can be led by the cloning site in the cloning vector of the present invention Enter foreign structural gene to be manufactured.To cloning site import foreign structural gene can be identical with the making of cloning vector make Use known method.

As long as being imported into the structural gene of the foreign structural gene coded protein of the expression vector of the present invention does not then have It is particularly limited to, can is the gene of the same race with the gene that the Schizosaccharomyces yeast as host has originally, may also be source In the structural gene of heterogeneity biological.By the structural gene (example using the encoding endogenous protein containing Schizosaccharomyces yeast Such as above-mentioned gsf2 genes) expression vector obtained by Schizosaccharomyces yeast transformant, can in a larger amount produce the endogenous Protein.Additionally, by using containing from heterogeneity biological structural gene expression vector obtained by Schizosaccharomyces yeast Transformant, substantial amounts of foreign protei matter can be produced.

The preferred foreign protei matter of protein coded by the foreign structural gene of the expression vector for being imported into the present invention, it is more excellent The protein being elected to be produced by the animal or plant of multicellular organism, what further preferred mammal (including mankind) produced Protein.In the case where such protein is manufactured using the prokaryotic such as Escherichia coli microbial hosts, mostly can not The high protein of activity is obtained, and in the case where the zooblasts such as Chinese hamster ovary celI are used as host, is typically produced Efficiency is low.Use the present invention expression vector, using Schizosaccharomyces yeast as host foreign protei matter expression system In the case of can solve these problems.

As long as it is imported into the gene of the foreign structural gene coded protein of the expression vector of the present invention, Ke Yishi The structural gene of wild type, or the gene for being changed the structural gene of wild type, or artificial synthesized Gene.As the structural gene beyond wild type, for example can enumerate coding merged wild type multiple protein it is chimeric The gene of protein, coding combine the gene of the protein of other peptides etc. on the N-terminal or C-terminal of wild-type protein Deng.As other peptide, secretion signal can be enumerated, to signal, the His marks such as transfer signal of specific intracellular organelle Labels such as label, FLAG labels etc..Various signals must be the signal worked in Schizosaccharomyces yeast.Secretion signal is logical Cross the peptide for being present in N-terminal and there is the function outside the Protein secretion to host cell for making to express.As in fission yeast The secretion signal worked in category yeast, the P3 signals described in particularly preferred International Publication No. 1996/23890.

[transformant and its manufacture method]

The transformant of second embodiment of the present invention is characterized in that the table of the second embodiment comprising the invention described above Up to carrier.The transformant of second embodiment of the present invention is carried out by importing above-mentioned expression vector in Schizosaccharomyces yeast Manufacture.

The host of the transformant of second embodiment of the present invention is Schizosaccharomyces yeast.Fragmentation used in the present invention Saccharomyces cerevisiae can be wild type, can also be the saltant type for making specific gene lack or inactivate according to purposes.As making spy The method for determining gene delection or inactivation, can adopt known method.Specifically, can be by using la tour method (Nucreic Acids Res, 2006, volume 34 the e11st, and described in International Publication No. 2007/063919 etc.) lacking gene Lose.Also can by using mutagens mutation partition method (molecular genetics in yeast experimental method, 1996, learn publishing centre), Random mutation method (PCR Methods Appl. (PCR method and application), 1992, volume 2, p.28-33.) using PCR etc. Mutation is introduced in a part for gene, the gene inactivation is thereby made.As the fission yeast for making specific gene lack or inactivate Category yeast host, such as in being recorded in International Publication No. 2002/101038, International Publication No. 2007/015470 etc..

Also, preferably use with the host for being used for the mark for selecting transformant as the Schizosaccharomyces yeast of host. For example, it is preferable to the host of specific nutrition composition is needed in growth using certain gene delection.Using comprising target gene sequence Carrier carry out being converted in the case of making transformant, by the gene (auxotrophy for integrating the disappearance in the carrier in advance Type complementary indicia), the auxotrophy for making the host of transformant disappears.By the host and transformant it is auxotrophic not Together, both can be distinguished, obtains transformant.As auxotroph complementary indicia, for example, can enumerate ura4 gene (auxotrophs Complementary indicia), isopropylmalate dehydrogenase gene (leu1 genes) etc..

The above-mentioned kind for enumerating can be utilized as the Schizosaccharomyces yeast that host uses.In above-mentioned Schizosaccharomyces yeast In, in terms of can be using various useful mutant strains, preferred schizosaccharomyces pombe.As grain wine used in the present invention The bacterial strain of fission yeast, for example, can enumerate ATCC38399 (leu1-32h-) or ATCC38436 (ura4-294h-) etc., these bacterium Strain can be obtained from American type culture collection (American Type Culture Collection).

Additionally, in the case of the expression vector of the foreign structural gene beyond importing with β-glucosyl enzym gene, Schizosaccharomyces pombe mutant in the first embodiment of the invention described above can be used as host.

The method for transformation of the Schizosaccharomyces yeast as host is converted using expression vector can be used known in any one Schizosaccharomyces yeast method for transformation.As such method for transformation, for example can enumerate lithium acetate method [K.Okazaki etc., Nucleic Acids Res., 18,6485-6489 (1990)], electroporation, protoplasm body, bead (glass Beads) method that in the past well-known method or Japanese Patent Laid-Open 2005-198612 publication were recorded such as method etc..Additionally, Also can be using commercially available yeast conversion kit.

After being converted, typically the transformant to obtaining is screened.Method as being screened, for example can example Lift method described below.Using the culture medium of transformant can be selected by above-mentioned nutrient defect type mark to be screened, from Multiple bacterium colonies are selected in the bacterium colony for obtaining.In addition, by carrying out to the transformant that these are selected based on pulsed field gel electricity The genome analysis of swimming method, can investigate out the carrier number or expression cassette number being incorporated in chromosome.

(cultural method)

The transformant of second embodiment of the present invention can be cultivated identically with natural Schizosaccharomyces yeast.As The cultural method, can enumerate the cultural method identical method with the transformant of above-mentioned first embodiment.Specifically, can make With the nutrient mediums such as YPD culture mediums (M.D.Rose etc., " Methods In Yeast Genetics (yeast geneticses sides Method) ", Cold Spring Harbor Labolatory Press (CSH Press) (1990)) or MB culture mediums Deng minimal medium (K.Okazaki etc., Nucleic Acids Res., vol.18, p.6485-6489 (1990)) etc..

Additionally, culture both can be batch culture, or fed-batch culture or continuous culture.

[production methods of exogenous proteins]

The method for producing protein of second embodiment of the present invention is characterized in that, implements to the second of the invention described above The transformant of mode is cultivated, and the egg coded by above-mentioned foreign structural gene is obtained from the thalline or nutrient solution supernatant for obtaining White matter.

Condition of culture is contemplated that produced species of target exogenous proteins etc. and is appropriately configured.For example with 16～ 42 DEG C, preferably 25～37 DEG C, carry out 8～168 hours, preferably 48～96 hours.Any one of shaken cultivation and quiescent culture is equal Can, can as needed be stirred or be breathed freely.

It is to have imported the conversion of the expression vector with hsp9 promoters in the transformant of second embodiment of the present invention In the case of body, if the transformant cultivated under conditions of the induction stimulation such as thermal pressure is given, hsp9 promoters Activated due to the pressure, the transcription of the foreign structural gene being controlled is promoted, and the exogenous proteins are expressed.At this In culture under induction incentive condition, compared with the culture under normal condition, the proliferative amount of usual Schizosaccharomyces yeast tails off. For this purpose, being cultivated with normal condition when culture starts, the cell concentration in nutrient solution has a certain degree of elevated Moment gives the induction such as thermal pressure to stimulate.Thereby, make cell quantitative change many by using the culture of early stage, as culture systems entirety Produce substantial amounts of foreign protei matter.Thermal pressure herein is one kind that induction stimulates, and by confirming various effects, induction stimulates Other methods can be used.As the method that induction stimulates, agent, peroxidating can be risen suitably by addition heat, cadmium, osmotic pressure Hydrogen, ethanol etc. are realized.

In the case of using heat, the temperature upper limit for giving thermal pressure is the highest temperature that Schizosaccharomyces yeast can survive Degree.Accordingly, as cultivation temperature during thermal pressure, be than giving thermal pressure before cultivation temperature be higher by preferably 2～20 DEG C, more It is preferred that 3～12 DEG C, most preferably 4～6 DEG C of temperature, and for 15～55 DEG C, preferably 25～45 DEG C, more preferably 30～40 DEG C.To giving The time for giving thermal pressure is not particularly limited, and effect can be confirmed more than several minutes, is 1～29 hour, it is appropriate that 1～15 Hour.

In the case where being added using cadmium, it is added in the form of cadmium ion.The ultimate density of cadmium is 0.1～1.5mM Till, till more preferably 0.5～1.0mM.What incubation time was adapted to is less than 5 hours, more preferably less than 3 hours.

In the case where agent is risen using osmotic pressure, the electrolyte or D-sorbite isosmoticity that can add high concentration rises Agent is improving osmotic pressure.It is more excellent till the ultimate density of potassium is 0.1～2.0M in the case of the potassium chloride using high concentration Till selecting 0.5～1.5M.Time to adding has no particular limits, it is appropriate that till 1～12 hour, and more preferably 1～10 Hour or so.

In the case of using hydrogen peroxide, till its ultimate density is 0.1～1.5mM, more preferably 0.5～1.0mM is Only.Time to cultivating has no particular limits, it is appropriate that till 1～15 hour, more preferably 1～12 hour or so.

In the case of using ethanol, till its ultimate density is 5～20V/V%, till more preferably 5～15V/V%.It is right The time of culture has no particular limits, it is appropriate that till 1～20 hour, more preferably 1～15 hour or so.

Above-mentioned condition can be processed individually or by multiple combinations.Combined effect can be by comparing expression easily Confirm.

It is to have imported the conversion of the expression vector with ihc1 promoters in the transformant of second embodiment of the present invention In the case of body, if starting to be cultivated in the state of cell density as time point is low in culture, exogenous proteins are not Considerably less amount is only expressed in expression.That is, because the transformant can be in the state of cell density be low with without foreign protein The state of the burden (or bearing little) of the expression of matter is bred, thus compared with the situation that there is burden, breeds efficiency high, Cell concentration can efficiently be increased.On the other hand, with the rising of cell density, expression is induced, as a result, can produce a large amount of Exogenous proteins.

After culture terminates, can be prepared containing target exogenous proteins by thalline by ultrasonic grind or mechanical crushing Cell extract, separates from the cell Extract, purifies exogenous proteins.Additionally, being secreted into cell in exogenous proteins In the case of outer, can separate from nutrient solution supernatant, purify exogenous proteins.As for obtaining the protein of these productions Separate, purification process, can enumerate it is known saltout or solvent precipitation etc. is using the other method of poor solubility, dialysis, ultrafiltration or Gel electrophoresis etc. utilize the other method of molecular weight differences, ion-exchange chromatography etc. to utilize charge difference method for distinguishing, affinity chromatography Method etc. using pathoklisis method, reversed-phased high performace liquid chromatographic etc. using hydrophobic gender gap method, isoelectric point electrophoresis Method etc. is using isoelectric point difference method for distinguishing etc..

As the method for protein for confirming to separate, purify, known immunoblotting or activation measurement can be enumerated Deng.The protein of purifying can be by clear and definite its structures such as amino acid analysis, amino terminal analysis, Primary Structure Analysis.

Embodiment

Embodiment and comparative example are illustrated below, the present invention is described in detail.But, the present invention is not exposed to following notes The restriction of load.

The making 1 of [test example 1] expression vector

By the peptide sequence of known AaBGL1, the base that design is replaced with the high expression type codon of schizosaccharomyces pombe Because of sequence (canonical sequence numbering 2.Hereinafter referred to as AaBGL1 genes).Add KpnI, BspHI identification sequences before initiation codon Row.Add XbaI, SacI recognition sequences after terminator codon.Restriction enzyme BspHI, XbaI enzyme cutting is used to contain the plasmid of the sequence (by GeneArt (ジーンアート) company, Regensburg, Germany synthesis).

On the other hand, restriction enzyme AarI, XbaI enzyme cutting pSL6lacZ is used to carry out alkaline phosphatase treatment in addition.In this place After reason, electrophoresis is carried out on Ago-Gel, carrier pSL6 fragments and above-mentioned AaBGL1 genes piece are cut out from Ago-Gel Section, after being attached (l igation), importing bacillus coli DH 5 alpha (precious biotech firm (タカラバイオ societies) system) carries out turning Change.Carrier is prepared by the transformant for obtaining, target expression vector pSL6AaBGL1 (Fig. 1, canonical sequence numbering 3) is obtained.It is logical Cross making restriction endonuclease map and confirm it is destination carrier.

Additionally, in order to be produced on N-terminal the AaBGL1 that with the addition of secretion signal P3 signals, using pSL6AaBGL1 as mould Plate is expanded using PCR methods using In-fusion primers to AaBGL1 genetic fragments.On the other hand, restriction enzyme AflII is used, XbaI carries out digestion to pSL6P3lacZ.In- is utilized by the fragment and by above-mentioned AaBGL1 genetic fragments obtained by PCR After fusion methods are cyclized, import bacillus coli DH 5 alpha and converted.Carrier is prepared by the transformant for obtaining, target is obtained Expression vector pSL6P3AaBGL1 (Fig. 2, canonical sequence numbering 4).By making restriction endonuclease map and confirming number of base sequence Confirmation is destination carrier.

The making 2 of [test example 2] expression vector

Additionally, in order to make using the AaBGL1 expression vectors of hsp9 promoters, making pSL6P3AaBGL1 as template With In-fusion primers P3AaBGL1 genetic fragments are expanded using PCR methods.On the other hand, restriction enzyme AarI, Xba1 are used Digestion has following pSL14lacZ of hsp9 promoters.By the fragment and by above-mentioned P3AaBGL1 genetic fragments obtained by PCR After being cyclized using In-fusion methods, import bacillus coli DH 5 alpha and converted.Carrier is prepared by the transformant for obtaining, Obtain target expression vector pSL14P3AaBGL1 (Fig. 3, canonical sequence numbering 5).By making restriction endonuclease map and confirming part It is destination carrier that base sequence confirms.

By hsp9 promoters (sequence number 6) with schizosaccharomyces pombe replace polyclonal carrier pSL9 (Figure 24, Promoter region (" inv1pro. " in Figure 24) 7038bp), makes polyclonal carrier pSL14 (Figure 25,6282bp).

Specifically, first, by from schizosaccharomyces pombe wild strain (ARC032 strains, ATCC38366, equivalent to Genomic DNA 972h-) uses the forward primer of the restriction enzyme sites for possessing SacI on 5 ' ends and 5 ' as template Possesses the reverse primer of the restriction enzyme sites of PciI on end, using PCR, to the ORF in the hsp9 genes of schizosaccharomyces pombe The region (sequence number 6) of 1～400bp of upstream of 5 ' ends (A of initiation codon ATG) expanded, obtain in the area Possess on the 5 ' ends in domain SacI, possess on 3 ' ends PciI restriction enzyme sites fragment.

Double digestion is carried out with restriction enzyme AarI and SacI to the startup subdivision of pSL9, and is attached, then converted After bacillus coli DH 5, plasmid is extracted, carry out the confirmation of base sequence.Although as a result, confirming the plasmid for obtaining in 3 ' ends It is upper to the addition of 1 adenine more, but with the promoter region comprising the base sequence represented by sequence number 6.By the plasmid It is denoted as pSL14.

Then, by being replaced with the region containing kalamycin resistance gene and pUCori, pSL14's is blue or green containing ammonia benzyl The region of mycin resistant gene and pBR322ori, integrates the structural gene of coding lacZ' on cloning site, makes many grams Grand carrier pSL14lacZ (Figure 26,6675bp, canonical sequence numbering 7).

The making (conversion to fission yeast) of [test example 3] transformant

Make the leucine auxotrophy strain (genotype of the schizosaccharomyces pombe as host：H-, leu1-32, university of Tokyo University Institute's Neo-Confucianism is that the wild male professor of research section subsidiary gene Experimental Establishment meal provides) (ATCC38399) in YES, (0.5% yeast is carried Take thing, 3% glucose, 0.1mg/mL SP supplements) 0.6 × 10 is grown in culture medium⁷Individual cell/mL.After collection, cleaning According to 1.0 × 10⁸The condition of individual cell/mL is suspended in 0.1M lithium acetates (pH5.0).Afterwards, add in the μ L of suspension 100 Carrier pSL14P3AaBGL1 obtained above is carried out into the μ g of product 1 of digestion with restriction enzyme SwaI, 50% (w/ is further added V) μ L of polyethylene glycol (PEG4000) aqueous solution 290 and after being sufficiently stirred for, successively with 5 minutes, room 60 minutes at 30 DEG C, at 42 DEG C The temperature order of lower 10 minutes is cultivated.PEG4000 is removed by centrifugation, during the aqua sterilisa of 150 μ L is suspended in after cleaning, coating In base agar culture medium.After 3 days, transformant is obtained (AaBGL1 expresses strain).By the transformant be denoted as ASP3660 strains (with Under, also referred to as usual strain).

The making of [test example 4] Δ pvg1 strains (pvg1 gene defect strains)

By uracil-deficient type strain (ARC010, the genotype of schizosaccharomyces pombe：H-leu1-32ura4-D18, Tokyo is big Xue great institutes Neo-Confucianism is that the wild male professor of research section subsidiary gene Experimental Establishment meal provides) according to la tour method (Nucreic Acids Res, 2006, e11 page of volume 34, described in No. 2007/063919 text of International Publication No.) converted, making is deleted Except the Δ pvg1 strains of pvg1 genes.The mutant that culture is obtained, carries out genome analysis, really using pulsed-field gel electrophoresis Recognize pvg1 gene defects.

In the making for deleting fragment, by the wild strain ARC032 strain (genotype based on schizosaccharomyces pombe：H-, Tokyo The big institute's Neo-Confucianism of university is that the wild male professor of research section subsidiary gene Experimental Establishment meal provides) by DNeasy (Kai Jie companies (キアゲ Application society) make) prepare complete genome DNA as template, carried out by PCR methods.

More specifically, fragment will be deleted and is divided into UP regions, OL regions and DN regions, by the DNA fragmentation in each region point Not by using KOD-Dash (Co., Ltd. (East oceans Spinning societies are spun by Japan) systems) PCR methods made after, further made For template, total length is made by identical PCR legal system and deletes fragment.

The making of [test example 5] Δ pvg1gsf2+ strains (pvg1 gene defect+gsf2 gene expressions increase strain)

Above-mentioned Δ pvg1 strains are made to cultivate in YES (0.5% yeast extract, 3% glucose, 0.1mg/mL SP supplements) 0.6 × 10 is grown in base⁷Individual cell/mL.According to 1.0 × 10 after collection, cleaning⁸The condition of individual cell/mL is suspended in 0.1M second In sour lithium (pH5.0).Afterwards, following genetic fragments containing ihc1 promoters and gsf2 genes are added in the μ L of suspension 100 (canonical sequence numbering 8) 1 μ g, further add the μ L of 50% (w/v) polyethylene glycol (PEG4000) aqueous solution 290 and are sufficiently stirred for Afterwards, successively with 60 minutes at 30 DEG C, 5 minutes at 42 DEG C, the room temperature order of lower 10 minutes cultivated.Removed by being centrifuged PEG4000, during the aqua sterilisa of 150 μ L is suspended in after cleaning, coats containing leucic base agar culture medium.After 3 days, can Obtain transformant (pvg1 gene defect+gsf2 gene expressions increase strain).FOA process is carried out afterwards so as to phonetic with urinating once again Pyridine deficiency.The mutant is denoted as into IGF799 strains.

The above-mentioned genetic fragment containing ihc1 promoters and gsf2 genes is made as follows.First, will be in ihc1 promoters The 5 '-end side of (canonical sequence numbering 9) contains gsf2 promoter sequences and Ura4 sequences, contains gsf2- in 3 '-end side The sequence of ORF expands said gene fragment as template using PCR.

The making (conversion to asexual flocculability fission yeast) of [test example 6] transformant

The above-mentioned IGF799 strains of the schizosaccharomyces pombe with asexual flocculability as host are made in YES (0.5% yeast Extract, 3% glucose, 0.1mg/mL SP supplements) 0.6 × 10 is grown in culture medium⁷Individual cell/mL.Collect, clean It is suspended in 0.1M lithium acetates (pH5.0) according to the condition of 1.0 × 108 cell/mL afterwards.Afterwards, add in the μ L of suspension 100 Entering, carrier pSL14P3AaBGL1 restriction enzymes SwaI obtained above are carried out the μ g of product 1 of digestion, further add 50% (w/v) μ L of polyethylene glycol (PEG4000) aqueous solution 290 and after being sufficiently stirred for, successively with 60 minutes at 30 DEG C, 5 minutes at 42 DEG C, The room temperature order of lower 10 minutes is cultivated.PEG4000 is removed by centrifugation, during the aqua sterilisa of 150 μ L is suspended in after cleaning, is applied It is distributed in the base agar culture medium containing uracil.After 3 days, transformant is obtained (AaBGL1 expresses strain).By the mutant note Make ASP4106 strains.

The making (supplementing uracil-deficient type) of [test example 7] transformant

(0.5% yeast extract, 3% glucose, 0.1mg/mL SP are mended in YES to make above-mentioned made ASP4106 strains Fill thing) 0.6 × 10 is grown in culture medium⁷Individual cell/mL.According to 1.0 × 10 after collection, cleaning⁸The condition of individual cell/mL is hanged In floating over 0.1M lithium acetates (pH5.0).Afterwards, add carrier pUC19-ura4 (Fig. 4, canonical sequence in the μ L of suspension 100 Numbering 10) the μ g of product 1 of digestion are carried out with restriction enzyme BsiwI, further add 50% (w/v) polyethylene glycol (PEG4000) water μ L of solution 290 and after being sufficiently stirred for, successively with 60 minutes at 30 DEG C, 5 minutes at 42 DEG C, the room temperature order of lower 10 minutes trained Support.PEG4000 is removed by centrifugation, during the aqua sterilisa of 150 μ L is suspended in after cleaning, base agar culture medium is coated.Fig. 4 In, the sequence of A and B represents the recombination site of carrier.After 3 days, transformant is obtained.This is supplemented into the urine of ASP4106 strains The transformant of pyrimidine deficiency is denoted as ASP4150 strains (hereinafter also referred to as flocculation strain).

The batch culture of [test example 8] transformant

The AaBGL1 expression strains (usual strain and flocculation strain) of above-mentioned gained are trained in test tube with YES culture mediums with 32 DEG C Support 24 hours.By nutrient solution 2mL in the YPD culture mediums (1% yeast extract, 2% peptone, 2% glucose) of 50mL Secondary Culture is carried out, formal culture in 48 hours is carried out with 32 DEG C in the conical flask of 500mL.

In order to evaluate the growing microorganism of transformant, light splitting light is used to the absorbance at the OD660nm of nutrient solution Degree meter (Spectrophotometer U-1500) is measured.In the case of the concentration height of nutrient solution stoste, using RO (Reverse Osmosis；Reverse osmosis) water is diluted and determines.When the OD660 of flocculation strain is determined, 100～500mM is suspended in EDTA, be measured after deflocculation.

The result of growing microorganism is shown in Fig. 5.In usual strain and flocculation strain, the rheological parameters' change with time of the OD660 values in culture Roughly the same change is shown as, final OD660 values are usual strain 23.4, strain 20.3 of flocculating.

Remaining concentration of glucose in nutrient solution and the concentration of alcohol as its metabolite are carried out with biology sensor BF5 Determine.The nutrient solution of collection is put into into microcentrifugal tube (eppendorf tube), is centrifuged using high speed micro centrifuge Separate, obtain culture supernatant.The culture supernatant 300 μ L are put in BF5 specimen cups, the BF5 for carrying out preparation of determine in advance is put into In automatic sampler.

BF5 is run under biology sensor service condition described below, is analyzed.

Using instrument：Biology sensor BF5 (prince's metrical instrument (prince's Meter Measuring Machine devices))

Flow velocity：1.0mL/ minute

Injection rate：5μL

Thermostat temperature：37℃

Dose times：90 seconds

Concentration quantitative method：The hydrogen peroxide for decomposing and producing due to the enzyme of glucose and ethanol is detected, with titer Obtain on the basis of peak height.

The result of the remaining concentration of glucose in nutrient solution is shown in Fig. 6 (a), used as the ethanol of the metabolite of the glucose The result of concentration is shown in Fig. 6 (b).In usual strain and flocculation strain, the Jing of concentration of glucose and concentration of alcohol in culture Shi Bianhua shows as roughly the same change.

Generally in strain and flocculation strain, the OD660 values in culture, the rheological parameters' change with time of concentration of glucose and concentration of alcohol exists Also change in an identical manner in each bacterial strain.Thus, thus it is speculated that under identical condition of culture, usual strain and flocculation strain are with substantially Same speed carries out the metabolism of sugar and the propagation of thalline.Think to give flocculability to growing microorganism using genetic manipulation Shadow affects little.

[test example 9] confirms the expression of AaBGL1 using determination of activity

The culture supernatant of strain is expressed using the AaBGL1 of above-mentioned gained, enzyme dilute sample is prepared, is carried out according to following methods Determination of activity.

(activation measurement)

In 20mM p-nitrophenyl-β-D- glucosides (below, being abbreviated as pNPG) 10 μ L, 1M sodium acetate buffers are added (pH4.5) the 10 μ L and μ L of water 130, the μ l of addition enzyme dilute sample 50, react 10 minutes at 37 DEG C.In 2% sodium carbonate liquor 100 The μ L of reactant liquor 100 are added to stop reaction in μ L, the amount of the p-nitrophenol under wavelength 450nm to dissociating carries out colorimetric and determines Amount.

The amount of the enzyme of p-nitrophenol equivalent to 1 μm of ol will be generated per 1 minute as 1U.Per 1mL usual strain and The pNPG degrading activities (active hereinafter also referred to as pNPG) of the culture supernatant in flocculation strain are shown in Fig. 7.

As shown in fig. 7, with regard to pNPG degrading activity values, culture 15.5 hours is strain of flocculating after glucose just exhaustion 1.38 times of activity values of usual strain are shown, after cultivating 48 hours, strain of flocculating shows 1.27 times of activity values of usual strain.

The fed-batch culture of [test example 10] transformant

In the usual strain of 5mL YES inoculation of mediums or flocculation strain, 24 hours precultures 1 are carried out with 32 DEG C in test tube. Further, the nutrient solution 4mL of the gained of preculture 1 is added in 200mL YES culture mediums, is carried out with 30 DEG C in 1L slope mouth flasks The preculture 2 of 24 hours.

Then, using 5L fermentation tanks, in the initial medium 1800mL that composition as shown in Table 1 is constituted preculture is added The nutrient solution 200mL of 2 gained, beginning is cultivated at 30 DEG C.Additionally, the concentration of each composition of table 1 represents that preculture 2 is inoculated with Concentration afterwards.After culture has started 14.0 hours, confirm that the remaining concentration of glucose in nutrient solution is less than 1.0g/L, start to mend Material.Afterwards, feed supplement 81 hours are continued, the supplemented medium 1450mL of the composition shown in feed supplement table 2 is cultivated 95 hours with 30 DEG C (representing the incubation time from culture starts).By the addition for controlling 12.5% ammoniacal liquor, pH is remained into 4.5.

[table 1]

Composition	Concentration
		Yeast extract	10g/L
Glucose (aqueous)	33g/L
		(NH₄)₂SO₄	15g/L
KH₂PO₄	8g/L
		MgSO₃·7H₂O	5.34g/L
Na₂HPO₄	0.04g/L
		CaCl₂·2H₂O	0.2g/L
Choline Chloride	15.00mg/L
		Folic acid	0.08mg/L
Pyridoxol	2.16mg/L
		Thin amine element	26.49mg/L
Thymidine	8.75mg/L
		Riboflavine sodium phosphate	7.97mg/L
P-aminobenzoic acid	38.16mg/L
		Calcium pantothenate	15.70mg/L
Nicotinic acid	49.00mg/L
		Inositol	27.00mg/L
Biotin	0.08mg/L
		FeC₆H₅O₇·nH₂O	28.40mg/L
ZnSO₄·7H₂O	25.40mg/L
		MnCl₂·4H₂O	4.10mg/L
CuSO₄·5H₂O	3.80mg/L
		H₂BO₃	2.90mg/L
Na₂MoO₄·2H₂O	0.68mg/L
		KI	0.20mg/L
NiSO₄·6H₂O	0.44mg/L
		CoCl₂·6H₂O	0.40mg/L

(glucose moisture content：8.8%)

[table 2]

	Concentration
		Yeast extract	10g/L
Glucose (aqueous)	550g/L
		CaCl_z2H₂O	0.20g/L
KH₂PO₄	9.00g/L
		MgSO₄7H₂O	4.45g/L
K₂SO₄	3.50g/L
		Na₂SO₄	0.14g/L
Na₂HPO₄	0.04g/L
		Choline Chloride	15.00mg/L
Folic acid	0.08mg/L
		Pyridoxol	2.16mg/L
Thin amine element	26.49mg/L
		Thymidine	8.75mg/L
Riboflavine sodium phosphate	7.97mg/L
		P-aminobenzoic acid	38.16mg/L
Calcium pantothenate	82.37mg/L
		Nicotinic acid	715.70mg/L
Inositol	693.70mg/L
		Biotin	0.74mg/L
FeC₆H₅O₇·nH₂O	473.4mg/L
		ZnSO₄·7H₂O	423.4mg/L
MnCl₂·4H₂O	68.4mg/L
		CuSO₄·5H₂O	63.4mg/L
H₂BO₃	48.4mg/L
		Na₂MoO₄·2B₂O	11.4mg/L
KI	3.4mg/L
		NiSO₄·6H₂O	7.4mg/L
CoCl₂·6H₂O	6.7mg/L

(glucose moisture content：8.8%)

In order to evaluate the growing microorganism of transformant, light splitting light is used to the absorbance at the OD660nm of nutrient solution Degree meter (Spectrophotometer U-1500) is measured.In the case of the concentration height of nutrient solution stoste, using RO water It is diluted and determines.When the OD660nm of flocculation strain is determined, the EDTA of 100～500mM is suspended in, deflocculation is laggard Row is determined.

The result of growing microorganism is shown in Fig. 8.In usual strain and flocculation strain, the rheological parameters' change with time of the OD660 values in culture Show as roughly the same change.Final OD660 values are usual strain 376, strain 395 of flocculating, by using will be used as deriving from day The semisynthetic medium that so yeast extract (Yeast Extract) of material is added in synthetic media realizes high thalline Concentration.

Remaining concentration of glucose in nutrient solution and as its metabolite concentration of alcohol with the identical side of test example 8 Method is measured.

The result of the remaining concentration of glucose in nutrient solution is shown in Fig. 9 (a), used as the ethanol of the metabolite of the glucose The result of concentration is shown in Fig. 9 (b).In usual strain and flocculation strain, the Jing of concentration of glucose and concentration of alcohol in culture Shi Bianhua shows as roughly the same change.Concentration of glucose after feed supplement starts is changed with the low value of below 1g/L. The ethanol produced in the stage of batch culture is reduced after feed supplement is started, and the concentration of alcohol in culture is finally with the low of below 1g/L Value is changed.In the alcohol production stage after starting as feed supplement to fed-batch culture terminates, there is no glucose Effect.

Generally in strain and flocculation strain, the OD660 values in culture, the rheological parameters' change with time of concentration of glucose and concentration of alcohol exists Change in an identical manner in each bacterial strain.Thus, thus it is speculated that under identical condition of culture, usual strain and flocculation strain are with substantially phase Same speed carries out the metabolism of sugar and the propagation of thalline.Think to give shadow of the flocculability to growing microorganism using genetic manipulation Affect little.

The measure of [test example 11] settling velocity

In 1L graduated cylinders to the fed-batch culture obtained by test example 10 at the end of each 1L of sample compare settling velocity. After making each graduated cylinder fully suspend, standing starts precipitation.By will from liquid surface to solid liquid interface (the yeast thalline of precipitation and The interface of supernatant) distance divided by precipitation start after elapsed time, calculate settling velocity.As a result it is shown in Figure 10.

As shown in Figure 10, confirm that compared with usual strain, flocculation strain settling velocity is fast.The settling velocity of flocculation strain is opened in precipitation Moment after beginning 2 hours is 30mm/ hours, and the moment after having spent 10 hours again (after precipitation starts 12 hours) is that 6.7mm/ is little When.On the other hand, moment of the settling velocity of usual strain after precipitation starts 12 hours is 1.5mm/ hours.

Additionally, Figure 11 represents microscopy observation result (Trypan Blue result) of the sample at the end of fed-batch culture. As shown in figure 11, by fed-batch culture at the end of sample microscopy observation results verification flocculation strain in substantial amounts of thalline set, The state of flocculation, by fed-batch culture, the flocculability of thalline does not disappear.Generally in strain, the wadding of thalline is not observed It is solidifying, in scattered state.

The optimization of [test example 12] cultivation temperature discusses (fed-batch culture)

In the usual strain of 5mL YES inoculation of mediums (ASP3660 strains), 24 hours are carried out in advance with 30 DEG C in L-type test tube Culture 1.Further, the nutrient solution 2.4mL of the gained of preculture 1 is added in 120mL YES culture mediums, in 500mL slope mouth flasks In carry out the preculture 2 of 24 hours with 30 DEG C.

Then, using 3L fermentation tanks, the nutrient solution 120mL of the gained of preculture 2 is added in initial medium 1080mL, is opened Beginning is cultivated.Respectively with 30 DEG C and 34 DEG C the two conditions enforcements of temperature conditionss in double-basis culture tank.Initial medium Composition uses removing yeast extract, Choline Chloride, folic acid, pyridoxol, thiamine, thymidine from the composition shown in table 1 The culture medium of deoxyribonucleoside, Riboflavine sodium phosphate and p-aminobenzoic acid.Additionally, the concentration of each composition of table 1 represents pre- training Support 2 postvaccinal concentration.Start feed supplement after starting 11.8 hours from culture.Afterwards, feed supplement 84.2 hours, the institute of feed supplement table 2 are continued The supplemented medium 685mL of the composition for showing, with 30 DEG C and 34 DEG C cultivate 96 hours (represents from culture beginning culture when Between).Supplemented medium also uses in the same manner removing yeast extract, Choline Chloride, folic acid, pyrrole from the composition shown in table 2 to tremble The culture medium of alcohol, thiamine, thymidine, Riboflavine sodium phosphate and p-aminobenzoic acid.By control The addition of 12.5% ammoniacal liquor, by pH 4.5 are remained.

In order to evaluate the growing microorganism of transformant, with the OD660nm's of the identical method of test example 8 measure nutrient solution Value.The result of growing microorganism is shown in Figure 12.At 30 DEG C and 34 DEG C, the rheological parameters' change with time of the OD660 values in culture shows as greatly Cause identical change.Final OD660 values are respectively 295 at 30 DEG C, are 271 at 34 DEG C, realize high cell concentration.

Remaining concentration of glucose in nutrient solution and as its metabolite concentration of alcohol with the identical side of test example 8 Method is measured.The result of the remaining concentration of glucose in nutrient solution is shown in Figure 13 A, used as the second of the metabolite of the glucose The result of determining alcohol is shown in Figure 13 B.At 30 DEG C and 34 DEG C, the Jing time-varying of concentration of glucose and concentration of alcohol in culture Change shows as roughly the same change.Concentration of glucose after cultivating 24 hours is changed with the low value of below 1g/L.Dividing Criticize the ethanol produced in culture to reduce after feed supplement is started, the concentration of alcohol in culture is finally become with the low value of below 2g/L Change.

PNPG degrading activities in nutrient solution with the identical method of test example 9 being measured.At 30 DEG C and 34 DEG C Figure 14 is shown in per the result of the pNPG degrading activities of 1mL culture supernatants.Culture 96 of the pNPG degrading activity values at the end of culture After hour, the activity value at 34 DEG C is shown as 2.26 times of the activity value at 30 DEG C.

[test example 13] is analyzed to identify the expression of AaBGL1 using SDS-PAGE

The each 5 μ L of culture supernatant sample at the end of the fed-batch culture of the gained of test example 12 are dissolved in into SDS-PAGE samples In savoring buffer solution, SDS-PAGE is carried out using 4-12% acrylamide gels, dyeed with Coomassie brilliant blue.The result of gained It is shown in Figure 15.The result of SDS-PAGE shows：After culture at the end of culture 96 hours, compared with 30 DEG C, at 34 DEG C, seen The band intensity for making the AaBGL1 of more than molecular weight 120kDa is strong, and the secreting, expressing amount of AaBGL1 increases.By on AaBGL1 Addition sugar chain, can confirm the band that trails.

[test example 14] is using the protein production of hsp9 promoters

Using the pEGFP-N1 containing GFP genes (CLONTECH companies (CLONTECH societies) system) as template, using In- Fusion primers expand the ORF fragments of EGFP gene by PCR methods.On the other hand, with restriction enzyme AflII and XbaI pair PSL14 carries out double digestion.In-fusion is utilized by the fragment of acquisition and by the ORF fragments of above-mentioned EGFP gene obtained by PCR After method is cyclized, imports bacillus coli DH 5 alpha and converted.Carrier is prepared by the transformant for obtaining, objective expression is obtained and is carried Body pSL14-EGFP (Figure 16).It is destination carrier by making restriction endonuclease map and confirming that number of base sequence confirms.

In the same manner, the carrier pSL6-EGFP (Figure 17) of the ORF fragments for incorporating EGFP gene is produced in pSL6, as Control.

Make the leucine auxotrophy strain (genotype of the schizosaccharomyces pombe as host：H-, leu1-32, university of Tokyo University Institute's Neo-Confucianism is that the wild male professor of research section subsidiary gene Experimental Establishment meal provides) (ATCC38399) in YES, (0.5% yeast is carried Take thing, 3% glucose, 0.1mg/mL SP supplements) 0.6 × 10 is grown in culture medium⁷Individual cell/mL.After collection, cleaning According to 1.0 × 10⁸The condition of individual cell/mL is suspended in 0.1m lithium acetates (pH5.0).Afterwards, add in the μ L of suspension 100 Expression vector pSL14-EGFP obtained above is carried out into the μ g of product 1 of digestion with restriction enzyme NotI, 50% (w/ is further added V) μ L of polyethylene glycol (PEG4000) aqueous solution 290 and after being sufficiently stirred for, successively with 5 minutes, room 60 minutes at 30 DEG C, at 42 DEG C The temperature order of lower 10 minutes is cultivated.PEG4000 is removed by centrifugation, during the aqua sterilisa of 150 μ L is suspended in after cleaning, coating In base agar culture medium.

The transformant obtained after 3 days is denoted as into ASP3395 strains.

In the same manner, import pSL14-EGFP and obtain the strain of transformant SL14E, import pSL6-EGFP and obtain transformant SL6E strains.

The ASP3395 strains for obtaining are seeded in the YES culture mediums of invisible spectro 5mL, are cultivated 70 hours at 32 DEG C.It is right Cultivate the fluorescence intensity in the case that the nutrient solution after terminating is excited under 488nm to be measured.

As control, SL14E strains and SL6E strains are cultivated at identical conditions, determine the nutrient solution after culture terminates Fluorescence intensity.

Measurement result is shown in Figure 18.As a result, using the fluorescence intensity of the nutrient solution of SL6E strains as in the case of 1, The fluorescence intensity (relative value) of SL14E strains is only 0.50, and the fluorescence intensity (relative value) of ASP3395 strains is very high, is 17.17。

The fluorescence intensity of nutrient solution is the index of the expression of fluorescin EGFP, it is known that hsp9 promoters are in fission yeast Expression efficiency is very high in category yeast, using transformant obtained by expression vector of the invention, with the feelings using other promoters Condition is compared, and can in a larger amount produce exogenous proteins.

[test example 15] is using the protein production of ihc1 promoters

The integrated recombinant vector pSL17 of single seat is made according to operation described below.I.e., first, by with grain wine fragmentation The promoter (ihc1 promoters) of the ihc1 genes of yeast replaces the known fission yeast polyclonal carrier pSL6 of recombinant type (figures 19,5960bp, sequence number 11) hCMV promoter regions, make polyclonal carrier pSL12 (Figure 20,5847bp).

Specifically, first, by from schizosaccharomyces pombe wild strain (ARC032 strains, ATCC38366, equivalent to Genomic DNA 972h-) uses the forward primer of the restriction enzyme recognition sequence for possessing BlnI on 5 ' ends as template (ihc1- promoter-F：With reference to table 3) and possess on 5 ' ends KpnI restriction enzyme recognition sequence reverse primer (ihc1- is opened Mover-R：With reference to table 3), using PCR, 5 ' end (initiation codons ATG to the ORF in the ihc1 genes of schizosaccharomyces pombe A) the region (sequence number 9) of 1～501bp of upstream expanded, obtain possessing on the 5 ' ends in the region BlnI, Possesses the fragment (ihc promoter fragments) of the restriction enzyme recognition sequence of KpnI on 3 ' ends.

In fragment after with restriction enzyme BlnI and KpnI double digestion pSL6, ihc is opened by connecting (ligation) Promoter fragment is incorporated into in the fragment after restriction enzyme BlnI and KpnI double digestions, obtains fission yeast integrating vector PSL12 (sequence number 14).

Then, the LPI ends of pSL12 are replaced by the terminator (ihc1 terminators) of the ihc1 genes with schizosaccharomyces pombe Only subregion, makes polyclonal carrier pSL17 (Figure 21,5831bp).

Specifically, first, by from schizosaccharomyces pombe wild strain (ARC032 strains, ATCC38366, relative to Genomic DNA 972h-) as template, using In-fusion primers (ihc1- terminator-F and ihc1- terminator-R：Reference Table 3) ORF in ihc1 genes using PCR to schizosaccharomyces pombe 3 ' ends (the 3rd letter of terminator codon) The region (sequence number 15) of 1～200bp of downstream is expanded, and obtains (the ihc terminators of the fragment containing ihc terminator regions Fragment).

Will be as pSL12 templates, using In-fusion primers (pSL12-F and pSL12-R：With reference to table 3) entered by PCR Row amplification, has obtained from defect in the total length of pSL12 the fragment of LPI terminator regions, and In-fusion gram is used in the fragment Grand kit (ProductName：In-Fusion HD Cloning Kit w/Cloning Enhancer, Takara Shuzo Co., Ltd.'s system) Integrate ihc and terminate sub-piece, make polyclonal carrier pSL17 (sequence number 20).

[table 3]

Using the pEGFP-N1 containing GFP genes (CLONTECH companies (CLONTECH societies) system) as template, using In- Fusion primers expand the ORF fragments of EGFP gene by PCR methods.On the other hand, with restriction enzyme AflII and XbaI to above-mentioned PSL12 carries out double digestion.Entered using In-fusion methods by the fragment and by the ORF fragments of above-mentioned EGFP gene obtained by PCR After row cyclisation, import bacillus coli DH 5 alpha and converted.Carrier is prepared by the transformant for obtaining, target expression vector is obtained PSL12-EGFP (Figure 22).It is destination carrier by making restriction endonuclease map and confirming that number of base sequence confirms.

In the same manner, the carrier pSL6-EGFP (Figure 17) of the ORF fragments for incorporating EGFP gene is produced in above-mentioned pSL6, As control.

Make the leucine auxotrophy strain (genotype of the schizosaccharomyces pombe as host：H-, leu1-32, university of Tokyo University Institute's Neo-Confucianism is that the wild male professor of research section subsidiary gene Experimental Establishment meal provides) (ATCC38399) in YES, (0.5% yeast is carried Take thing, 3% glucose, 0.1mg/mL SP supplements) 0.6 × 10 is grown in culture medium⁷Individual cell/mL.After collection, cleaning According to 1.0 × 10⁸The condition of individual cell/mL is suspended in 0.1m lithium acetates (pH5.0).Afterwards, add in the μ L of suspension 100 Expression vector pSL12-EGFP obtained above is carried out into the μ g of product 1 of digestion with restriction enzyme NotI, 50% (w/ is further added V) μ L of polyethylene glycol (PEG4000) aqueous solution 290 and after being sufficiently stirred for, successively with 5 minutes, room 60 minutes at 30 DEG C, at 42 DEG C The temperature order of lower 10 minutes is cultivated.PEG4000 is removed by centrifugation, during the aqua sterilisa of 150 μ L is suspended in after cleaning, coating In base agar culture medium.

The transformant obtained after 3 days is denoted as into 277G strains.

In the same manner, pSL6-EGFP is imported, obtains the strain of transformant SL6E.

The 277G strains for obtaining are seeded in the YES culture mediums of invisible spectro 5mL, are cultivated 72 hours at 32 DEG C.To from Culture starts to after culture terminates, the suction of fluorescence intensity and 600nm in the case of the exciting under 488nm of nutrient solution Luminosity to enter determined when passing through.

As control, SL6E strains are also cultivated at identical conditions, the fluorescence intensity and 600nm to nutrient solution Absorbance enter when passing through determine.

Figure 23 shows " fluorescence intensity/OD600 " of the nutrient solution of each strain, and (fluorescence intensity is divided by the absorbance of 600nm Value) rheological parameters' change with time.As a result, " fluorescence intensity/OD600 " of 277G strains (being in figure " ihc1p ") is starting Jing from culture It is lower than the SL6E strains (being in figure " hCMVp ") using hCMV promoters to spend moment of 24 hours, but is beginning to pass through 48 from culture Than SL6E plant height after hour, the expression of the EGFP of per unit yeast is high.

Specification, the right of the Japanese patent application 2012-181865 that in August, 2012 incorporated herein is filed an application on the 20th Announcement of the full content of claim, accompanying drawing and summary as the specification of the present invention.

Claims

1. a kind of cloning vector, it is characterised in that possess：It is made up of the base sequence represented with sequence number 9 and with startup The promoter of sub- activity, positioned at the downstream of the promoter and by promoter control for import foreign structural gene gram Grand site, and the terminator that can be worked in Schizosaccharomyces yeast.

2. cloning vector as claimed in claim 1, it is characterised in that the promoter is schizosaccharomyces pombe 1～the 501bp of upstream of the 5 ' ends of the ORF of the ihc1 genes of (Schizosaccharomyces pombe), i.e. ORFs Region.

3. a kind of manufacture method of expression vector, it is characterised in that the clone in the cloning vector described in claim 1 or 2 Foreign structural gene is imported on site.

4. a kind of expression vector, it is characterised in that import on the cloning site in the cloning vector described in claim 1 or 2 There is foreign structural gene.

5. a kind of manufacture method of the transformant of Schizosaccharomyces yeast, it is characterised in that carry the expression described in claim 4 Body imports Schizosaccharomyces yeast.

6. a kind of transformant of Schizosaccharomyces yeast, it is characterised in that containing the expression vector described in claim 4.

7. a kind of manufacture method of protein, it is characterised in that the transformant described in culture claim 6, from the thalline for obtaining Or obtain protein coded by the foreign structural gene in nutrient solution supernatant.